PRACTICAL 


HISTOLOGY  AND  PATHOLOGY 


BY 

HENEAGE  GIBBES,   M.B. 


LONDON: 

fl.  K.  LEWIS,  136  GOWER  STREET,  W.C. 
1880. 


PREFACE. 


THE  object  of  this  small  work  is  to  lay  before  the 
practitioner  and  student  of  medicine,  a  few  concise 
and  simple  methods,  by  which  the  various  tissues  of 
the  body  may  be  prepared  for  examination  with  the 
microscope. 

I  do  not  claim  any  originality  in  these  methods,  but 
I  recommend  them  from  my  own  personal  experience 
as  the  best,  easiest,  and  cheapest  to  carry  out. 

The  use  of  dyes  for  staining  the  tissues  is  a  com- 
paratively new  branch  of  the  science  of  Histology,  and 
its  value  is  being  proved  every  day. 

I  have  been  engaged  in  experiments  with  all  the 
various  colouring  agents  for  a  long  time,  and  in  trying 
their  effect  in  double  and  treble  staining,  and  I  have 
given  the  result  of  those  which  have  proved  most  suc- 
cessful hitherto.  I  have  also  added  a  list  of  the  aniline 
dyes,  those  soluble  in  water,  and  those  soluble  in  spirit, 
which  will  be  found  very  useful  by  anyone  wishing  to 
make  experiments  in  staining. 


iv  Preface. 

Briefly,  but  sincerely,  I  offer  my  best  thanks  to 
Dr.  Klein,  F.E.S.,  for  the  assistance  which  he  has  at 
all  times  freely  and  generously  given  me. 

H.  G. 

94  Gower  Street. 

October  1,  1880. 


TABLE  OF  CONTENTS, 


CHAPTEE  I. 

INTRODUCTION. 

PAGE 

The  Microscope  ...  .  .4 

Achromatic  Condenser     .  . 

On  large  stands  for  high  powers        .  .  .  . 

On  the  Binocular  Microscope       .  .  9 

CHAPTEE  II. 

ON   PREPARING   TISSUES   FOR   EXAMINATION. 

Chromic  Acid  Mixture            .            .            .            .  .            .11 

Muller's  Fluid .12 

Dilute  Spirit     ...                       .  ,     13 

Bichromate  of  Potash        .....  13 

Chromate  of  Ammonia            .            .            .            .  .            .13 

Chloride  of  Gold    .......  14 

Picric  Acid       ......  .15 

CHAPTEE  III. 
ON  CUTTING  SECTIONS. 

Freezing  Microtome          ......  18 

To  prepare  material  for  freezing        .            .            .            .  .19 

To  make  Mucilage            .           .           .           .            .           .  20 

Cutting  the  Sections  .           .           .           .           .           •  .20 


vi  Contents. 


CHAPTER  IV. 
ON  STAINING. 

PAGE 

List  of  Staining  Agents     .....  22 

Logwood  Stain             ...                       .  .23 
On  Staining  Sections  with  Logwood  that  have  been  prepared 

with  Chromic  Acid     ....  24 

Carmine            -  .24 

Indigo-carmine       •                                   •  25 

Purpurin           .  25 

Anthra-purpurin    •  26 

Eosin      .  •    26 

Picro-carmine         •  27 
Anilin  Dyes 
Anilin  Dyes  soluble  in  Spirit 


CHAPTEE  V. 
ON  DOUBLE  STAINING. 

Picro-carmine  and  Logwood   . 

Carmine  and  Indigo -carmine  .                                                         37 

Treble  Staining             .  -    39 

Chloride  of  Gold  and  Anilines  •                                                        42 


CHAPTER  VI. 
ON  MOUNTING. 

Slides     ....  44 

Cover  Glasses         .  44 

On  measuring  Cover  Glasses  •  45 

On  cleaning  Cover  Glasses  45 


Contents.  vii 


PAGE 

Mounting  Fluids          ...  .    46 

To  mount  in  Canada  Balsam        .  .           .                       .          48 

On  breaking  down  old  preparations   .  .                        -52 

Mounting  Large  Sections  ...                      53 

Thin  Slides        -                                  .  .    55 


CHAPTEE  VII. 

Method  of  obtaining  Animal  Tissues  for  Examination             .  56 

Dissection  of  Frog        •  .    58 

Dissection  of  Newt            ......  59 


PRACTICAL  HISTOLOGY. 

Blood     .  .....    60 

Epithelium  .  •  «...          62 

Cartilage  .  ...    69 

Bone  .  «...          71 

Muscular  Tissue  .  .  .  .  .  -72 

Nervous  Structures  75 

Blood  Vessels   .  .....    79 

Salivary  Glands,  Pancreas  ....          81 

Teeth      .  82 

Alimentary  Canal  .  ...          82 

Liver      .  •  .  .  .  .  .  .  .84 

kung  .          86 

Kidney   .  ....    86 

Genital  Organs,  Male        .....  33 

Genital  Organs,  Female  ....  -90 

Spermatozoa  •  •  •  .  .          90 

Special  Senses  •  .  .  .  .  .  .  .93 

Nasal  Organ  .  .  -95 

Eye         .......  .95 


viii  Contents. 


PRACTICAL  PATHOLOGY. 

PAGE 

On  preparing  and  mounting  Pathological  Specimens    .            .  99 

To  make  permanent  preparations  of  a  Cancer  in  a  short  time  •    99 

On  Double  and  Treble  Staining  Morbid  Growths         •            •  101 

Large  Sections  of  Pathological  Specimens    .            .  •  101 

Amyloid  Degeneration      .                       .                                   .  102 

Hydatids            •  .  102 
Short  history  of  the  manner  in  which  a  portion  of  the  Morbid 

Growth  is  prepared  by  the  Chromic  Acid  method            •  103 


PEACTICAL  HISTOLOGY  AND  PATHOLOGY. 


CHAPTEE  I. 

INTRODUCTION. 

HISTOLOGY  or  the  minute  anatomy  of  healthy  tissue  as 
revealed  by  the  microscope,  has  made  vast  strides  in 
the  last  few  years,  and  now  forms  a  most  important 
part  of  Medical  education ;  a  thorough  knowledge  of 
the  normal  structure  of  the  animal  tissues  being  ab- 
solutely necessary  for  the  appreciation  of  pathological 
change. 

This  subject  is  now  taught  at  the  Medical  schools, 
and  there  is  no  excuse  for  a  student  who  has  finished 
his   curriculum   without   some   knowledge  of  practical 
histology,  and  of  the  minute  anatomy  of  the  human, 
body. 

There  are  many,  however,  who  qualified  some  years 
ago  when  little  attention  was  paid  to  this  subject,  who 
have  consequently  no  practical  knowledge  of  the  methods 
required  to  prepare  any  morbid  growth  they  may  meet 
in  their  professional  career,  or  how  to  set  about  a  micro- 
scopical examination  of  the  same. 

With  a  view  to  help  those  who  have  not  been  able 
to  get  the  necessary  knowledge  during  their  student 
career,  and  also  those  students  who  wish  to  form  a 
Laboratory  at  home,  the  present  work  has  been  written, 


Introduction. 


giving  only  the  ordinary  methods  used  by  the  author  in 
his  own  Laboratory. 

When  all  these  methods  have  been  thoroughly  worked 
out,  the  student  will  find  himself  competent  to  try  any 
of  the  various  processes  mentioned  in  larger  works,  and 
to  judge  of  their  utility. 

Many  men  on  reading  the  different  hardening,  cut- 
ting, staining,  and  mounting  processes  which  any  tissue 
has  to  undergo  before  it  can  be  examined  with  the 
microscope,  will  be  inclined  to  think  it  very  tedious 
work.  It  is,  however,  a  mere  matter  of  routine,  and 
when  once  this  routine  is  established,  the  whole  thing 
is  comparatively  simple.  It  takes  very  little  time  to 
change  the  hardening  fluid,  and  if  the  student  gets  into 
the  habit  of  looking  over  the  bottles  on  the  shelf  every 
morning,  where  he  keeps  those  tissues  in  the  process  of 
hardening,  a  glance  at  the  labels  will  show  those  re- 
quiring a  change.  When  the  sections  are  mounted  and 
examined  under  the  microscope,  he  will  find  himself 
amply  repaid  for  all  his  trouble  if  he  has  faithfully 
carried  out  the  different  processes  in  every  detail. 

It  is  always  better  to  have  one  or  two  shelves  devoted 
to  those  preparations  which  require  changing ;  and  those 
such  as  chromic  acid,  which  require  fresh  fluid  often, 
should  be  kept  by  themselves.  Each  bottle  should  be 
labelled,  and  the  tissue,  date,  and  hardening  fluid, 
clearly  written  on  the  label.  Every  morning  this 
shelf  should  be  examined,  and  those  requiring  it, 
changed;  the  date  being  each  time  written  on  the 
label,  so  that  it  may  be  seen  at  a  glance  how  long  the 
tissue  has  been  in  the  fluid,  and  whether  the  hardening 


Introduction. 


agent  ought  to  be  renewed.  Miiller's  fluid,  and  bichro- 
mate of  potash  preparations,  may  be  placed  by  them- 
selves, and  need  only  be  looked  at  occasionally. 

A  large  outlay  is  not  required  for  a  course  of  Histo- 
logical  investigation,  and  the  following  list  will  show  all 
those  articles,  re-agents,  &c.,  which  the  student  will  find 
absolutely  necessary. 

1.  Microscope.    1  Eye-piece.     2  Object  glasses. 

2.  |  gross  ground  edge,  3+1  slides. 

3.  1  oz.  No.  1,  f  square  cover  glasses. 

4.  1  oz.  No.  1         „         „          „ 

5.  1  hollow  ground  razor. 

6.  Needles  in  handles. 

7.  2  pair  sharp  pointed  forceps. 

8.  1  pair  broad  pointed  forceps,  not  roughed,  for  taking  up  clean 

cover  glasses. 

9.  Copper  lifter. 

EE-AGENTS. 

f  per  cent.  Salt  solution. 
\      ,,         Solution  Nitrate  of  Silver. 
I      „  „        Chloride  of  Gold. 

5      ,,  ,,        Bicarbonate  of  Soda. 

Glycerine  "\ 

Canada  Balaam        >  In  drop  bottles. 

Dammar  Yarnish     J 

A  Bottle  of  Hollis'  Glue. 
A  Williams'  Microtome. 
Watch  Glasses. 
Glass  Capsules. 
Dissecting  Case. 
Curved  Scissors. 

B2 


The  Microscope. 


THE  MICKOSCOPE. 

THE  most  expensive  as  well  as  the  most  important  arti- 
cle required  is  the  microscope,  and  a  good  one  should 
be  procured. 

In  purchasing  a  microscope,  it  is  necessary  to  goto 
an  optician  who  makes  his  own  instruments  to  get  one 
that  is  worth  anything,  as  a  large  number  are  made  by 
wholesale  manufacturers  and  sold  to  various  traders 
who  put  their  names  on  them  and  sell  them  as  their 
own  make.  It  is  an  easy  matter  to  find  out  a  bona 
fide  maker,  as  there  are  very  few  of  them,  and  it  is  their 
interest  to  sell  a  good  instrument.  A  microscope 
such  as  that  required  by  anyone  beginning  a  course  of 
histology,  can  be  procured  for  £5  5s.,  and  nothing  worth 
having  can  be  bought  at  a  lower  price.  If  the  instru- 
ment be  procured  from  a  bona  fide  maker  it  will  last  a 
lifetime  with  ordinary  care. 

The  compound  microscope  consists  of  the  stand,  eye- 
piece, and  object  glasses.  The  stand  should  be  a  tripod 
having  a  stage  of  blackened  glass,  and  a  draw-tube 
lined  with  cloth.  The  tripod  foot  gives  perfect  steadi- 
ness, and  for  this  reason  the  Continental  models  with 
horse- shoe  foot  have  been  given  up  by  many  of  the  best 
microscopists. 

The  price  of  these  stands,  with  two  object  glasses, 
ranges  from  £5  5s.  to  £6  12s.  6d. 

The  student  should  ascertain  by  looking  down  the 
tube  with  the  eye- piece  removed,  whether  the  hole  in 
the  stage  is  concentric  with  the  tube,  and  then  try  the 
different  holes  in  the  diaphragm  in  the  same  way.  The 


The  Microscope. 


diaphragm  is  an  important  part  of  the  stand,  as  it  is 
very  often  necessary  to  cut  off  some  of  the  rays  of 
light  to  get  the  best  effect  with  high  powers  ;  and  for 
this  purpose  the  tubular  diaphragm  which  is  slipped 
into  a  fitting  under  the  stage,  is  the  best;  it  has  a 
very  small  hole,  and  can  be  removed  altogether  when 
using  a  low  power ;  a  good  plan  is  to  have  no  fixed  dia- 
phragm at  all,  but  a  small  piece  of  metal  with  a  milli- 
metre hole  in  the  centre,  made  so  that  it  can  be  slipped 
into  the  hole  in  the  stage  from  above,  where  it  will  lay 
flush  with  the  glass  plate.  It  must  be  carefully  cen- 
tred with  the  tube. 

Having  selected  a  maker,  the  student  should  get  him 
to  explain  the  working  of  the  different  parts,  as  all  that 
is  necessary  for  a  beginner  can  be  learnt  in  ten  minutes 
in  that  way. 

This  small  student's  stand  is  all  that  will  be  required 
by  the  student  for  a  long  time,  as  with  it  high  powers 
can  be  used  and  whatever  work  may  be  done  in  the 
future,  it  will  always  be  the  working  stand. 

Good  students'  microscopes  are  made  by  Mr.  Crouch, 
66  Barbican;  Messrs.  Swift  and  Son,  University  Street. 

The  Object  Glass.  The  object  glass  or  objective,  is 
the  most  important  part  of  the  microscope,  and  it  is 
necessary  to  have  good  glasses  to  do  satisfactory  work. 

The  most  useful  for  the  student  are  the  f  or  |-  for  a 
low  power,  and  the  J,  1,  or  ^  for  a  high  power ;  with 
a  f  and  ^  the  student  can  do  all  the  requisite  work  for 
a  long  time,  and  then  with  the  addition  of  a  J,  and  oil 
immersion  ^  he  would  be  set  up  for  life. 

The  student  should  get  some  good  microscopist  to 
test  the  object  glasses  for  him  before  purchasing  them, 


The  Microscope. 


and  he  should  see  that  they  are  tested  on  some  histo- 
logical  object,  and  not  on  diatoms,  as  the  wide  angle 
necessary  for  resolving  test  diatomaceae  are  the  reverse 
of  useful  to  the  young  Histologist.  The  most  important 
glass  for  the  student  is  the  high  power,  and  it  is  neces- 
sary that  this  should  be  a  good  one. 

High  Powers.  The  best  high  powers  are  made  by 
Messrs.  Powell  and  Lealand,  they  are  of  course  expen- 
sive but  pay  well  in  the  end;  they  are  made  with 
correction  collars  and  are  more  adapted  to  large  stands 
with  mechanical  stage.  Zeiss'  E  =  j-  and  F  =  ^  are 
very  good  glasses,  and  well  adapted  to  the  students' 
stand  and  are  not  expensive ;  they  have  no  correction 
collar,  and  the  cover  glasses  should  be  measured  to  get 
the  best  effect ;  -006  will  do  very  well  for  D  and  E,  and 
•004  for  F,  the  same  applies  to  Mr.  Crouch's  ±  which  is 
corrected  to  a  cover  glass  of  -006. 

Oil  Immersion  Lenses.  These  glasses  are  taking  the 
place  of  water  immersion  high  powers  in  Histologi- 
cal  research,  as  they  have  no  correction  for  thickness  of 
cover  glass,  and  are  consequently  much  easier  to  use, 
the  only  drawback  is  that  the  essential  oil  used  will 
dissolve  Canada  balsam,  Dammar  varnish,  and  many  of 
the  other  sealing  fluids,  and  it  is  necessary  to  cover  them 
with  Hollis'  glue  which  is  not  acted  on  by  cedar  oil. 

These  glasses  were  first  made  by  Zeiss  of  Jena,  and 
since  by  Messrs.  Powell  and  Lealand.  The  first  glass 
made  by  Zeiss  was  the  J-,  he  then  brought  out  a  T^- 
and  afterwards  a  J^,  of  these  the  T^  is  unquestionably 
the  best  glass.  Messrs.  Powell  and  Lealand  have  made 
_3^?  _!_,  _i_y  and  -^3-  on  this  principle,  and  have  succeeded 
in  removing  a  great  objection  to  oil  immersion  lenses  ; 


The  Microscope. 


they  have  made  their  glasses  perfectly  homogeneous, 
that  is,  they  require  no  change  of  the  oil  when  using 
oblique  light  on  an  object,  and  no  correction  with  the 
draw  tube  is  necessary  when  using  a  very  thin  cover. 
With  Zeiss'  -^  it  is  necessary  to  draw  out  the  tube  two 
inches  with  a  '004  cover  glass,  and  for  central  light  a 
mixture  of  fennel  and  olive  oils  is  required,  which  must 
be  changed  to  cedar  oil  for  oblique  light. 

With  Messrs.  Powell  and  Lealand's  glasses  cedar  oil 
is  used  indifferently  for  central  or  oblique  light,  and  no 
correction  is  necessary  for  a  thick  or  thin  cover  glass. 

In  using  oil  immersions,  all  that  is  required  is  to 
place  a  very  small  drop  of  oil  on  the  front  lens,  screw 
the  glass  into  the  body,  and  lower  it  on  to  the  slide  until 
contact  is  made,  which  can  be  seen  by  bringing  the  eye 
to  a  level  with  the  surface  of  the  slide.  The  glass  is 
then  focussed  by  the  fine  adjustment  until  the  object  is 
seen  sharply  defined.  With  these  glasses  the  best  effect 
is  obtained  the  moment  the  glass  is  in  focus,  and  they 
have  an  enormous  superiority  over  a  dry  glass  of  the 
same  power  in  sharpness  of  definition  and  brilliancy. 
All  slides  intended  for  use  with  high  powers  should  be 
sealed  with  Hollis'  glue,  and  then  the  cedar  oil  left  on 
the  cover  glass  can  be  wiped  off  without  the  least  dan- 
ger to  the  preparation. 

Eye- Pieces.  Students'  microscopes  are  generally  sold 
with  two  eye-pieces,  nos.  1  and  3,  or  A  and  C,  but  the 
no.  1  or  A  is  the  only  one  required,  as  no  good  result  is 
obtained  by  using  an  eye -piece  with  a  higher  magnify- 
ing power,  the  higher  eye-piece  only  magnifying  the 
image  seen  with  the  object  glass. 


Achromatic  Oondensor. 


Higher  eye-pieces  are,  however,  useful  in  testing  an 
object  glass,  as  very  many  of  those  sold,  while  giving 
fairly  good  results  with  a  no.  1  eye-piece,  become  blurred 
and  indistinct  when  a  no.  3  or  4  is  used. 

Illumination.  Daylight  is  the  best  light  to  use  for 
Histological  work,  and  in  the  summer  time  there  is 
generally  enough  light  for  ordinary  work.  In  the  win- 
ter, however,  a  lamp  is  often  required  even  in  the  day- 
time, as  there  are  many  days  when  sufficient  light 
cannot  be  obtained. 

It  is  necessary,  therefore,  to  be  provided  with  a  lamp 
burning  either  gas  or  parafine  oil.  The  best  gas  lamp 
is  made  by  Baker,  it  is  a  modification  of  the  Highley 
microscopic  lamp  and  is  fitted  with  a  Sugg's  burner,  it 
will  be  found  very  useful.  An  ordinary  parafine  lamp 
which  can  be  purchased  for  2s.,  with  a  f  inch  flat  wick, 
will  be  found  all  that  is  necessary  by  those  who  do  not 
wish  to  get  the  more  expensive  gas  lamp.  It  has  also 
this  advantage,  the  wick  being  flat  the  edge  of  the  flame 
can  be  turned  to  the  microscope,  giving  an  intensity  of 
light  very  useful  in  the  investigation  of  fine  structures 
under  very  high  powers. 

ACHKOMATIC  CONDENSER. 

A  SMALL  condenser  made  to  go  into  the  fitting  under  the 
stage  will  be  found  very  useful,  but  for  high  power  work 
with  a  large  stand  a  wide  angled  condenser  is  necessary, 
having  a  series  of  stops  for  oblique  light,  and  a  graduated 
diaphragm  by  which  the  rays  of  light  can  be  gradually 
cut  off  until  the  best  definition  is  obtained. 


On  Large  Stands  for  High  Powers.  9 

STAND  CONDENSEK. 

It  is  better  to  use  the  direct  light  of  the  lamp  without 
the  intervention  of  a  stand  condenser,  as  by  this  instru- 
ment the  light  is  diffused  and  the  sharpness  of  definition 
impaired.  A  stand  condenser  is  required  to  throw  a 
light  on  opaque  preparations,  but  as  these  are  not  often 
used  by  the  student  of  Histology,  it  is  not  required  by  a 
beginner. 

ON  LARGE   STANDS  FOR  HIGH  POWERS. 

THE  advanced  student  will  require  a  Stand  with  me- 
chanical stage  and  sub- stage  when  he  makes  investi- 
gations into  minute  structures  with  high  powers,  and 
for  this  work  it  is  most  important  that  the  mechanism 
of  the  Stand  should  be  of  the  very  best  description.  To 
get  this  he  must  go  to  the  best  makers,  and  of  course  be 
prepared  to  pay  a  high  price. 

The  best  Stands  are  made  by  Messrs.  Powell  and 
Lealand,  and  Messrs.  Boss  and  Co. 

The  author  has  used  a  No.  2  stand  of  Messrs.  Powell 
and  Lealand  for  a  long  time,  and  found  it  all  that 
could  be  desired. 

ON  THE  BINOCULAR  MICROSCOPE. 

THE  binocular  microscope  has  not  met  with  much  favor 
from  Histologists  as  yet,  from  the  fact  that  no  power 
above  a  %  inch  could  be  used  without  a  special  stand  or 
apparatus  costing  a  large  sum.  As,  however,  it  has 
been  shown  by  the  author*  that  the  binocular  can  be 

*  See  Quarterly  Journal  of  Microscopical  Science  for  July,  1880. 


10  On  the  Binocular  Microscope. 

used  with  the  T^  oil  immersion,  the  student  should  try 
it,  and  see  what  a  different  view  it  gives  of  various 
structures  and  their  relation  to  one  another.  Any  or- 
dinary stand  made  on  the  Jackson -Lister  model  will  do, 
so  that  the  body  can  be  brought  almost  into  contact 
with  the  stage.  A  Zeiss'  D  can  be  made  to  work  per- 
fectly well  by  fitting  the  front  part  into  an  adapter,  so 
as  to  bring  the  lenses  close  to  the  prism.  The  relief 
to  the  eyes  is  very  great. 

For  a  higher  power  Messrs.  Powell  and  Lealandmake 
their  T^  oil  immersion  with  a  screw  cut  on  the  outside, 
so  that  the  front  part  containing  the  lens  can  be  screwed 
into  an  adapter,  which  they  supply  with  the  glass.  By 
this  means  perfect  stereoscopic  effect  is  obtained,  and 
the  observer  is  enabled  to  realise  the  precise  relations  of 
the  different  structures  he  is  looking  at  to  one  another. 


CHAPTEE  II. 

ON  PEEPABING  TISSUES  FOE  EXAMINATION. 

THE  most  essential  point  in  microscopic  investigation  is 
the  proper  hardening  of  the  material  to  be  examined, 
and  this  must  be  done  gradually,  as  if  any  tissue  is 
placed  in  a  strong  solution,  the  elements  of  which 
it  is  composed  at  once  shrink,  and  it  is  impossible  to 
form  any  correct  idea  of  their  nature. 

1.  Chromic  Acid  Mixture.  The  most  useful  harden- 
ing agent  is  a  mixture  of  chromic  acid  and  spirit.  Make 
a  solution  of  chromic  acid  in  water  15  grains  to  the 
pint,  this  is  about  ^  per  cent.  Take  of  this  2  parts ; 
methylated  spirit  1  part. 

The  material  must  be  cut  into  small  pieces  about  half 
an  inch  square,  and  a  large  quantity  of  fluid  used,  a  wide 
mouthed  stoppered  bottle  holding  from  6 — 10  ozs.,  ac- 
cording to  the  quantity  of  material,  is  best ;  change  the 
fluid  at  the  end  of  24  hours,  and  again  every  third  day, 
and  the  material  will  be  hardened  in  from  8 — 12  days, 
this  can  be  easily  proved  by  taking  out  a  piece  and  feel- 
ing it.  If  allowed  to  remain  too  long  it  gets  brittle. 
When  it  is  found  to  be  moderately  hard,  usually  after 
about  9 — 10  days,  pour  off  the  chromic  acid  mixture, 
and  wash  well,  replace  it  by  dilute  spirit  made  thus : — 

Take 

Methylated  Spirit  2  parts,  and 

Water  1  part. 


12          On  Preparing  Tissues  for  Examination. 

Let  the  material  remain  in  this  for  from  24 — 36  hours, 
never  longer  than  three  days,  and  then  replace  it  by 
pure  methylated  spirit,  it  may  remain  in  this  for  an  in- 
definite time,  but  it  will  often  be  found  that  the  spirit 
becomes  cloudy  and  full  of  deposits  in  a  few  days ;  in 
this  case  it  is  only  necessary  to  change  the  spirit  until 
it  remains  clear. 

A  large  quantity  of  i  per  cent,  solution  of  chromic 
acid  should  be  kept  on  hand,  and  it  should  be  mixed 
with  the  spirit  as  required,  it  will  be  found  the  most 
useful  of  all  the  hardening  agents,  if  it  is  changed  at 
the  proper  times,  and  it  should  not  be  used  stronger 
than  is  given  above,  it  will  even  be  found  beneficial  to 
use  it  weaker  in  some  cases. 

2.  Muller's  Fluid  is  a  good  hardening  mixture,  but 
requires  a  much  longer  time,  taking  weeks  to  do  what 
the  chromic  acid  mixture  will  do  in  days.  It  is  made 
thus : — 

Take 

Potass.  Bichrom.  2  parts. 

Sodse  Sulph.  1  part. 
Water  100  parts. 

The  advantage  of  this  mixture  is  that  larger  pieces 
can  be  hardened  in  it,  and  it  does  not  require 
changing  after  the  first  week  or  two,  but  it  will  take 
from  5 — 7  weeks  to  harden  anything  according  to 
its  size.  The  material,  when  sufficiently  hardened, 
should  be  well  washed  and  then  placed  in  dilute  spirit 
in  the  same  manner  as  recommended  after  hardening 
in  the  chromic  acid  mixture. 


On  Preparing  Tissues  for  Examination.         13 

3.  Dilute  Spirit.      Many  tissues  can  be  hardened  in 
spirit  alone  if  they  are  placed  in  dilute  spirit  at  first, 
so  that  the  elements  of  which  they  are  composed  are 
not  shrunk.      This  process  is  also  used  after  hardening 
by  any  of  the  others. 

Dilute  spirit  is  made  by  adding  1  part  of  water  to 
2  parts  of  methylated  spirit. 

The  material  to  be  hardened  should  not  be  left  in 
this  mixture  more  than  from  24 — 48  hours. 

It  is  then  transferred  to  pure  methylated  spirit. 

4.  Bichromate  of  Potash.     Make  a  2  per  cent,  solution 
and  keep  it  on  hand,  as  it  is  very  useful  for  many  tis- 
sues that  require  slow  hardening.     A  solution  can  be 
made  much  more  quickly  with  warm  water  than  cold. 
This  solution  is  also  very  useful  to  place  portions  of 
morbid  material  in,  on  their  removal  from  the  body  in 
the  post-mortem  room,  they  can  afterwards  be  trans- 
ferred to  the  chromic  acid  mixture  for  more  rapid  hard- 
ening.    This  solution  takes  from  three  to  seven  weeks 
to  harden  according  to  the  size  of  the  specimen,  and  the 
frequency  with  which  the  solution  is  changed. 

5.  Bichromate  of  Ammonia.    A  2  per  cent,  solution  is 
used  in  precisely  the  same  manner  as  the  former,   and 
is  applicable  to  the  same  tissues. 

6.  Chr ornate  of  Ammonia.     Make  a  5  per  cent,   solu- 
tion, that  is  1  oz.  of  the  salt  to  20  oz.  of  water,  and 
filter.     Keep  it  in  a  stoppered  bottle.     When  this  harden- 
ing agent  is  used  for  fresh  tissue,  such  as  mesentery,  a 
small  quantity  is  placed  in  a  glass  vessel  and  the  tissue 
immersed  in  it  for  24  hours,  it  is  then  washed  until  no 
more  colour  comes  away  and  mounted  in  glycerine. 


14         On  Preparing  Tissues  for  Examination. 

For  other  tissues  it  is  necessary  that  the  material 
should  be  cut  into  small  pieces  and  left  in  the  solution 
for  24 — 48  hours.  It  is  then  placed  in  distilled  water, 
which  must  be  changed  several  times  until  it  is  no 
longer  tinged.  The  hardening  is  completed  by  the  spirit 
process  (page  13). 

7.  Chloride  of  Gold.  Half  per  cent,  solution.  This  is 
sold  in  small  glass  tubes,  each  containing  15  grains  of 
the  chloride,  equal  to  7  grains  of  pure  gold.  Take  one 
of  these  tubes  and  file  a  ring  round  it  above  the  bulb, 
it  can  then  be  easily  divided  into  two  parts,  empty  the 
gold  chloride  into  a  6- ounce  bottle,  and  wash  out  any 
particles  that  remain  with  distilled  water,  fill  up  the 
bottle.  This  will  be  under  a  half  per  cent,  solution,  but 
answers  very  well.  Place  a  small  quantity  of  this  solu- 
tion in  a  watch  glass  and  immerse  the  tissue,  which 
must  be  perfectly  fresh,  in  it,  let  it  remain  in  the  dark 
for  from  half  to  one  hour  or  more,  then  place  in  dis- 
tilled water,  which  must  be  changed  several  times,  and 
expose  to  diffuse  daylight  until  it  becomes  a  violet 
brown ;  about  24  hours  will  do  in  summer. 

The  tissue  can  then  be  mounted  in  glycerine,  if  it  is 
a  small  thin  substance,  such  as  a  Tadpole's  tail. 

If,  however,  larger  portions  of  any  tissue  are  stained 
with  gold  chloride,  the  hardening  will  not  be  sufficient, 
and  they  must  be  further  hardened  by  the  spirit  pro- 
cess (page  13). 

Mouse- tail,  stained  and  hardened  by  the  gold  process, 
may  be  decalcified  by  placing  it  in  a  half  per  cent,  solu- 
tion of  chromic  acid  or  a  saturated  solution  of  picric 
acid  for  a  few  days. 


On  Preparing  Tissues  for  Examination.         15 

For  another  gold  process,  see  Cornea.  Gold  chloride 
has  a  staining  as  well  as  a  hardening  action. 

8.  Picric  Acid.     A  saturated  solution  of  picric  acid 
will  decalcify  small  bones.      It  is   also  used  in  some 
cases  as  a  hardening  agent  by  adding  1  part  of  water 
to  2  parts   of  a   saturated   solution ;    but  it  does  not 
seem  to  give   such  good  results  as  the  chromic  acid 
mixture. 

9.  Osmic  Acid.    This  can  be  procured  as  a  1  per  cent, 
solution  in  water,   and  it  is  then   diluted  to  various 
strengths  as  required.     It  blackens  fat  and  the  medul- 
lary sheath  of  nerves. 

A  piece  of  mesentery  placed  in  a  weak  solution  for 
half  an  hour  will  show  the  fat  cells  lying  along  the 
course  of  the  blood-vessels,  as  round  black  bodies. 

It  is  also  used  for  hardening  the  internal  ear. 


CHAPTEE  III. 
ON   CUTTING  SECTIONS. 

SECTIONS  may  be  cut  either  by  hand  with  a  razor,  or 
with  a  microtome. 

1.  In  cutting  sections  by  hand  it  is  necessary  to  im- 
bed the  tissue  in  some  material  which  will  cut  easily, 
and  at  the  same  time  hold  it  firmly. 

The  best  substance  for  this  purpose  is  a  mixture  of 
wax  and  olive  oil. 

Take  equal  parts  of  white  wax  and  olive  oil  by  weight 
and  melt  them  together,  pour  into  a  shallow  vessel  and 
when  cold  cut  into  small  blocks. 

Small  tin-boxes  with  a  removable  bottom  are  re- 
quired to  hold  the  mixture  while  the  tissue  is  being  im- 
bedded, and  the  best  size  is  2  inches  long,  by  f  of  an 
inch  wide,  and  J  of  an  inch  deep. 

It  will  be  necessary  also  to  have  a  small  porcelain 
ladle  and  a  stand  to  raise  it  above  a  spirit  lamp  or  gas 
jet.  Melt  some  of  the  wax  mass  in  the  ladle  and  be 
careful  not  to  make  it  too  hot. 

Prepare  the  tissue  so  that  the  face  which  is  to  be  cut 
can  be  easily  recognised,  stick  a  needle  into  it  away 
from  the  part  which  is  to  be  cut,  drain  off  most  of 
the  spirit  by  laying  it  on  filtering  paper,  and  then  im- 
merse in  the  melted  wax  mass,  so  that  it  is  perfectly 
covered,  take  it  out  and  let  it  cool.  Take  a  small  piece 
of  filter  paper  and  place  it  over  the  removable  bottom 


On  Cutting  Sections.  17 

of  the  tin  box,  and  then  fix  it  in  its  place,  the  filter 
paper  will  prevent  the  wax  from  running  out  if  the  bot- 
tom fits  loosely.  It  is  also  useful  to  leave  a  little  of  the 
paper  projecting  on  which  to  write  the  name  of  the 
material  imbedded.  Then  half  fill  the  box  with  melted 
wax  mass  and  hold  the  material  in  it,  keeping  it  quite 
steady  until  the  wax  hardens,  then  by  gently  screwing 
the  needle  round  it  can  easily  be  removed,  and  the  box 
filled  up  with  wax  mass.  It  will  be  found  a  saving  of 
time  to  imbed  a  portion  of  material  at  each  end  of  the 
box.  When  the  wax  mass  has  become  thoroughly 
hard,  which  will  take  some  time,  especially  in  warm 
weather,  pull  off  the  bottom  and  push  the  wax  mass, 
with  the  filter  paper  adhering  out  of  the  box.  It  can 
then  be  laid  by  until  wanted,  the  name  of  the  material 
imbedded  being  written  on  the  paper. 

For  cutting  sections  thus  imbedded,  a  hollow  ground 
razor  is  necessary ;  a  very  good  one  for  this  purpose  can 
be  procured  from  Baker,  High  Holborn ;  the  razor  must 
be  very  sharp.  A  small  glass  capsule  about  J  of  an 
inch  deep  filled  with  methylated  spirit  is  also  required 
to  put  the  sections  into  when  cut,  and  to  moisten  the 
razor  in. 

Take  the  wax  mass  and  with  a  scalpel  carefully 
remove  small  slices  from  one  end,  until  the  imbedded 
mass  can  just  be  seen,  then  take  the  razor  and  dip  it 
into  the  capsule,  taking  up  a  little  spirit,  let  this  run 
along  the  edge  so  as  thoroughly  to  moisten  it,  and  com- 
mence cutting  as  thin  sections  as  possible,  by  drawing 
the  razor  diagonally  across  the  mass  with  a  steady 
sweep ;  this  must  not  be  done  too  quickly,  and  the 

c 


18  The  Freezing  Microtome. 

amount  of  pressure  to  be  put  on  the  razor  will  depend 
on  the  tissue  imbedded  and  can  only  be  learnt  by  prac- 
tice. As  each  section  is  cut  dip  the  razor  into  the  cap- 
sule of  spirit  and  wash  it  off.  Wipe  the  razor  occa- 
sionally and  remove  adhering  portions  of  wax  mass, 
and  always  keep  the  edge  wetted  with  spirit.  When  a 
sufficient  number  of  sections  have  been  cut,  the  thin- 
est  should  be  selected  and  removed  to  a  watch  glass 
containing  clear  spirit. 

Great  care  is  required  in  cutting  sections  by  hand,  to 
hold  the  razor  firmly  yet  lightly,  so  as  to  cut  them  thin 
and  at  the  same  time  even,  and  this  cannot  be  done 
without  a  great  deal  of  practice. 

For  larger  sections  the  boxes  must  be  proportionately 
increased  in  size,  and  it  will  be  found  convenient  when 
the  wax  mass  is  as  wide  or  wider  than  the  razor  to  cut 
off  slices  from  each  side  so  as  to  reduce  the  surface  to 
be  cut  as  much  as  possible  without  interfering  with  the 
stability  of  the  imbedded  material. 

A  small  flat  spear-headed  needle  will  be  found  useful 
for  taking  up  very  small  sections. 

THE  FREEZING  MICROTOME. 

A  much  easier  method  of  cutting  sections  is  by  using 
a  microtome.  Of  these  there  are  a  number  made,  in 
some  of  which  the  material  is  imbedded  in  wax  mass, 
or  a  mixture  of  paraffin  and  lard,  and  raised  gradually 
by  a  screw,  while  a  razor  is  worked  on  a  flat  plate 
shaving  off  sections  ;  these  are  also  made  with  a  cham- 
ber to  contain  a  freezing  mixture,  so  that  the  material 
can  be  imbedded  and  frozen. 


To  prepare  the  Material  for  Freezing.  19 

Only  one  microtome  will,  however,  be  described  in 
this  work,  as  it  is  very  simple  and  does  the  work  well. 
This  is  Williams'  microtome,  made  by  Swift  of  Univer- 
sity Street.  It  consists  of  a  tub  to  contain  the  freezing 
mixture,  with  a  brass  standard  into  which  screw  the 
brass  circular  plates  on  which  the  material  is  frozen. 
A  top  with  a  glass  surface  fits  on  to  this,  having  a  hole 
through  which  the  circular  plate  projects.  The  knife 
is  fixed  into  a  triangular  frame,  having  screws  at  each 
angle  by  which  it  is  raised  and  lowered. 

To  prepare  the  microtome  for  use  : 

1.  Have  the  knife  as  sharp  as  possible. 

2.  Pound  some  ice  finely  in  a  cloth. 

3.  Scrape  some  salt  into  a  fine  powder. 

With  a  spoon,  put  a  layer  of  ice  into  the  tub  and  then 
some  salt  on  it,  mix  with  the  spoon,  and  so  on,  until  the 
tub  is  about  half  full,  then  ram  it  hard  with  a  stick  and  fill 
again,  put  in  the  salt  and  ice  in  about  equal  proportions, 
leave  room  for  the  top,  wipe  off  the  salt  and  ice  from 
the  edge,  put  on  the  top  and  fix  it  with  the  screw  for 
that  purpose.  Screw  the  circular  plate  into  its  place, 
and  the  microtome  is  ready  for  use. 

An  India-rubber  tube  must  be  fixed  to  carry  away 
the  drainings  as  the  ice  melts. 

To  PREPAKE  THE  MATERIAL  FOR  FREEZING. 

Any  tissue  which  has  been  preserved  in  spirit, 
must  be  soaked  in  water  for  24  hours  to  remove  the 
spirit,  and  then  placed  in  mucilage  for  another  24 

hours. 

c2 


20  Cutting  the  Sections. 

It  will  be  found  a  great  saving  of  time,  when  a  num- 
ber of  specimens  are  to  be  cut  at  one  freezing,  not  to 
have  the  material  too  thick,  as  a  piece  a  quarter  of  an 
inch  thick  will  give  an  enormous  number  of  thin  sec- 
tions, and  take  only  a  short  time  to  freeze. 

To  MAKE  MUCILAGE. 

Pour  warm  water  on  picked  gum  Acacia  and  make  a 
solution  rather  thicker  than  the  mucilages  sold  in  the 
shops. 

CUTTING  THE  SECTIONS. 

Eemove  all  the  specimens  to  be  cut,  from  the  gum, 
and  place  them  in  a  small  saucer  ready  at  hand.  Take 
up  one  with  a  pair  of  forceps  and  lay  it  on  the  circular 
plate  of  the  microtome,  drop  some  gum  solution  on  it 
with  a  small  brush,  and  see  that  it  runs  down  on  to  the 
plate  all  round  the  specimen  so  as  to  fix  ib  firmly. 
When  it  is  thoroughly  frozen  adjust  the  razor  so  that 
ib  will  just  pass  over  without  touching,  and  then  lower 
the  screw  at  the  apex  of  the  triangle  by  giving  it  a 
slight  turn  to  the  left  and  push  the  knife  across  the 
material  in  a  diagonal  direction.  Have  a  small  vessel 
ready  containing  warm  distilled  water.  It  is  necessary 
to  use  disbilled  water,  as  in  ordinary  water  the  lime  in 
solution  is  precipitated  by  boiling  and  the  specimen  will 
be  covered  by  fine  particles  of  carbonate  of  lime  and 
utterly  ruined.  Moisten  the  upper  surface  of  the  razor 
with  gum  solution,  and  the  sections  as  they  are  cut  will 
slip  up  on  it  without  curling,  carefully  remove  them 
with  a  camel-hair  brush  and  place  them  in  the  warm 


Cutting  the  Sections.  21 

distilled  water  and  let  them  remain  for  10  minutes  or 
longer,  until  the  gum  is  dissolved  out ;  this  will  take 
longer  with  some  material,  such  as  testicle,  than  others. 

With  this  microtome  the  most  beautiful  sections  can 
be  cut  perfectly  even  throughout,  surpassing  anything 
that  can  be  done  with  the  hand.  With  care  very  large 
sections  may  be  cut  quite  as  readily  as  smaller  ones, 
but  to  cut  them  well  the  razor  must  be  very  sharp,  and 
the  material  not  too  hard ;  those  hardened  in  chromic 
acid  mixture,  No.  1  (page  11)  seem  to  do  the  best. 
Very  little  force  is  required  in  pushing  the  knife  across 
the  material,  and  if  it  is  sharp  a  very  slight  turn 
of  the  screw  each  time  will  cut  a  section  ;  these  ought 
to  be  so  thin  as  to  be  almost  invisible,  as  the  gum  melts 
on  the  razor. 

In  cutting  some  material,  such  as  retina,  it  is  advis- 
able to  stain  it  en  masse  before  freezing,  otherwise  the 
sections  cannot  be  seen  when  placed  in  water. 


CHAPTEE  IV. 

ON  STAINING. 
LIST  OP  STAINING  AGENTS. 

1.  Hsematoxylin  or  Logwood  5.  Anthra-purpurine 

2.  Carmine  6.  Eosin 

3.  Indigo-czvrmine  7.  Picro- carmine. 

4.  Purpurine 

Aniline  Dyes  Soluble  in  Water. 

8.  *Soluble    Aniline    or    China        13.  Dahlia,  Eosanilin   and  Me- 

Blue  thylanilin  Violet 

9.  *Pure  Soluble  Blue  14.  Malachite  Green 

10.  Serge  Blue  15.  *lodine  Green 

11.  Tyrian  Blue  ]  6.  *Bismarck  Brown. 

12.  *Safranine 

Aniline  Dyes  Soluble  in  Spirit. 

17.  Spiller's  Purple  21.  Citranine 

18.  *Rosein  22.  Aurine 

19.  Anilin  Red  23,  *Pure  Opal  Blue. 

20.  *Anilin  Violet 

Selective  Stains. 
Osmic  Acid  Chloride  of  Gold  Nitrate  of  Silver. 

To  demonstrate  the  minute  structure  of  any  tissue,  the 
sections  require  to  be  stained  with  some  colouring 
agent  which  will  show  the  different  elements  more 
plainly  by  their  absorption  of  the  colouring  matter  and 
bring  out  very  transparent  parts  which  otherwise  would 
be  hardly  discernible. 

Of  all  the  staining  agents,  logwood  is  the  most  useful, 
and  a  solution  prepared  in  the  following  manner  will  be 
found  the  most  efficient. 


To  Stain  with  Logwood.  23 

LOGWOOD  STAIN. 
1.  Take  of 

Extr.  Hsematoxyl,  grms.  6 
Alumen,  grms.  18. 

Mix  thoroughly,  while  mixing  add  28  c.c.  of  distilled 
water.  Filter  and  add  to  the  filtrate  3  j  of  spirit  of 
wine.  Keep  in  a  stoppered  bottle  a  week  before  using. 
What  remains  on  the  filter  can  be  mixed  with  14  c.c.  of 
distilled  water,  and  left  soaking  in  it  for  an  hour  or  so, 
then  filter  and  add  to  the  filtrate  3  BS  of  spirit  of  wine. 
This  second  solution  is  as  strong  as  the  first. 

To  STAIN  WITH  LOGWOOD. 

Make  a  cone  with  a  small  round  filter  paper,  and 
pour  some  of  the  staining  fluid  into  it,  let  from  seven  to 
ten  drops  fall  into  a  watch  glass  and  dilute  with  dis- 
tilled water.  Let  the  sections  remain  in  the  solution  for 
about  a  quarter  of  an  hour,  the  time  will  depend  on  the 
tissue  and  the  manner  in  which  it  has  been  hardened. 
Some  tissues  take  in  the  stain  very  rapidly,  others 
slowly.  Take  out  a  section  from  time  to  time,  and  place 
it  in  a  watch  glass  of  ordinary  filtered  water  to  see  if  it 
is  stained  deeply  enough. 

When  the  sections  appear  to  have  stained  thoroughly 
remove  them  to  a  watch  glass  of  ordinary  water,  and 
wash  them  to  remove  the  excess  of  colouring  matter. 
In  staining  with  logwood  it  is  necessary  to  be  careful 
that  too  many  sections  are  not  placed  in  the  solution 
at  once,  as  they  will  lie  thickly  one  on  the  other,  and 
the  staining  will  not  be  uniform  ;  it  is  also  necessary  to 


24  On  Staining. 


dilute  the  logwood  stain  with  distilled  water,  as  or- 
dinary water  will  not  give  the  same  result,  owing  to 
the  different  matters  held  in  solution ;  but  it  is  better 
to  use  ordinary  water  for  washing  the  sections  after 
staining  as  it  helps  to  fix  the  color. 

The  solution  should  not  be  too  strong,  as  better  re- 
sults are  obtained  from  staining  the  sections  slowly 
than  from  doing  it  rapidly,  and  it  will  always  result 
in  a  loss  of  time  if  an  attempt  is  made  to  stain  a  large 
number  of  sections  in  a  strong  solution. 

ON  STAINING  SECTIONS    WITH  LOGWOOD  THAT  HAVE  BEEN 
PREPARED  WITH  CHROMIC  ACID. 

It  is  necessary  to  remove  the  chromic  acid  from  sec- 
tions hardened  in  that  fluid,  and  this  is  done  by  taking 
some  of  the  5  per  cent,  solution  of  bicarbonate  of  soda 
diluting  it  slightly,  and  soaking  the  sections  in  it  for 
some  time,  ten  to  twenty  minutes  will  generally  be  suf- 
ficient. 

Then  remove  them  to  plain  water  and  wash  well. 

Prepare  two  watch  glasses  of  dilute  logwood  stain 
and  place  the  sections  in  one  of  them,  let  them  remain 
for  a  minute  and  then  place  them  in  the  other ;  there 
they  must  stay  until  stained  deeply  enough. 

If  the  first  watch  glass  be  now  examined,  the  log- 
wood stain  will  be  found  to  have  become  quite  granular, 
and  if  this  precaution  had  not  been  taken  it  would  have 
been  deposited  as  minute  granules  all  over  the  sections. 

2.  Carmine.  This  stain,  formerly  so  much  used,  is 
now  almost  given  up  by  Histologists,  as  a  general  stain, 
in  favor  of  logwood  which  is  found  to  differentiate  the 


On  Staining.  25 


tissues  much  better  while  it  has  not  the  deleterious 
effect  of  carmine  on  the  eyes  when  used  with  lamp 
light. 

Carmine  is,  however,  valuable  in  double  staining, 
either  as  a  carmine  solution  or  in  conjunction  with 
picric  acid  as  picro- carmine. 

The  carmine  solution  is  prepared  by  mixing : 

Carmine,  3  ss. 
Borax,  3  ij. 
Aqua,  ^  iv. 

and  pouring  off  the  clear  supernatant  fluid.  It  must 
not  be  filtered. 

3.  Indigo- Carmine  or  Sulphindigotate  of  Soda.     This 
is  a  useful  stain  in  conjunction  with  carmine,  especially 
for  pathological  specimens. 

First  make  a  saturated  solution  of  the  powder  in  dis- 
tilled water  and  filter,  take  some  of  this  solution  and 
pour  it  into  methylated  spirit  until  it  has  attained  a 
moderately  deep  color ;  a  good  deal  of  the  colouring 
matter  will  be  precipitated,  and  it  must  be  carefully 
filtered  to  remove  this,  it  is  then  ready  for  use.  The 
solution  should  not  have  a  very  deep  blue  color  and 
when  held  to  the  light,  should  show  a  purplish  tint.  It 
does  not  require  long  to  stain  the  sections  in  it,  and 
they  should  be  allowed  to  remain  long  enough  to  stain 
them  evenly,  as  it  will  be  found  that  they  stain  first  at 
the  edges,  after  having  been  passed  through  dilute  hy- 
drochloric acid. 

4.  Purpurine.      True   purpurine   is  a  red  colouring 
matter  extracted  from  madder,  it  is  rather  expensive 


26  On  Staining. 


and  not  easily  obtained.  It  is  soluble  in  spirit  with 
the  addition  of  slight  heat. 

Place  some  of  the  powder  in  a  test  tube  and  add 
some  rectified  spirit,  warm  gently  over  a  spirit  lamp. 
When  dissolved,  filter  and  keep  in  a  stoppered  bottle. 
It  stains  readily  and  brings  out  some  tissues  such  as 
muscle  and  mucous  glands  well. 

It  does  very  well  in  double  staining  with  aniline 
blues. 

5.  Anthra-Purpurine.       A   brown   powder   which   is 
often  sold  for  purpurine.     It  is  isomeric  with  it,  but 
is  found  as  a  secondary  product  in  the  preparation  of 
alizarin  from  anthracene. 

It  is  very  slightly  soluble  in  water  and  may  be  re- 
moved entirely  from  aqueous  solutions  by  means  of 
ether. 

A  solution  may  be  made  by  first  dissolving  it  in 
spirit  and  then  adding  a  warm  solution  of  alum  in 
water,  the  color  will  then  become  reddish.  It  stains 
muscle  and  epithelium  but  not  connective  tissue. 

It  does  not  show  up  minute  structures  as  well  as 
logwood. 

It  does  not  combine  with  picro- carmine  in  double 
staining,  but  the  difference  in  the  two  colors  is  so  slight 
as  to  make  this  of  little  value. 

6.  Eosin.     Is  not  an  anilin  color  but  is  a  potash  salt 
of  resorcin.      Kesorcin   is  obtained  by  the   action  of 
melting  potash  on  galbanum.    Eosin  is  largely  soluble 
in  water  and  has  a  beautiful  garnet  red  color.     In  using 
it  a  strong  solution  is  required  and  the  sections  must 
be  well  washed  in  water  after  staining.    It  is  a  very 


On  Staining.  27 


delicate  color  when  used  alone  and  almost  too  trans- 
parent, but  in  combination  it  becomes  opaque. 

This  is  the  ordinary  eosin,  there  is  also  an  alcoholic 
eosin  called  primrose  which  is  much  used  by  silk  dyers, 
it  is  soluble  in  a  weak  solution  of  spirit  of  wine,  it  fixes 
readily  and  gives  a  more  brilliant  color  than  ordinary 
eosin,  and  is  not  affected  by  light.  There  is  also  a  blue 
shade  of  eosin  which  is  said  to  have  a  great  affinity  for 
wool,  and  can  be  fixed  readily  by  a  solution  of  soda 
hypophosphis ;  this  might  be  useful  for  investigations 
in  diseases  of  the  hair. 

7.  Picro-  Carmine.  This  useful  stain  is  difficult  to 
make  and  the  process  is  very  tedious,  it  is  better  there- 
fore to  buy  it  prepared  for  use.  It  can  be  procured 
from  Martindale,  New  Cavendish  Street,  at  a  reason- 
able price. 

Filter  about  10  drops  into  a  watch-glass  and  dilute 
with  distilled  water.  The  sections  must  remain  for 
some  time,  from  twenty  minutes  to  half  an  hour,  and 
if  at  the  end  of  that  time  they  have  not  stained  suffi- 
ciently, a  little  more  picro-carmine  may  be  added. 

They  are  then  placed  in  water  acidulated  with  a  few 
drops  of  acetic  or  picric  acid  and  left  for  an  hour. 

When  making  experiments  in  treble  staining  a  num- 
ber of  sections  may  be  stained  in  picro-carmine  and 
then  placed  in  methylated  spirit ;  there  they  may  re- 
main until  required,  as  the  spirit  does  not  affect  the 
stain,  which  forms  a  very  good  ground  color,  on  which 
to  try  combinations  of  different  anilines. 

It  is  also  a  good  stain  for  fresh  tissues,  such  as 
mesentery  when  used  with  logwood.  Also  in  sections  of 


28  On  Staining. 


skin  it  is  very  useful  as  a  ground  color,  and  by  staining 
a  number  of  sections  of  the  same  tissue  with  picro- 
carmine  and  then  with  two  other  colors,  a  variety  of 
results  will  be  obtained. 

In  some  the  tissue  for  which  picro- carmine  has  a 
special  affinity,  such  as  connective  tissue  will  be  found 
unaltered,  while  the  surrounding  tissue  has  taken  on 
the  new  colors.  In  others  the  picro- carmine  has  com- 
bined with  one  or  both,  and  a  new  color  is  formed. 
Or  again,  it  may  be  entirely  supplanted  by  one  of  the 
other  colors. 

ANILIN  DYES. 

These  may  be  divided  into  two  classes,  those  soluble 
in  water  and  those  soluble  in  spirit ;  and  this  classifica- 
tion will  be  found  very  useful  for  experiments  in  double 
staining. 

There  are  an  immense  number  of  anilin  dyes  now 
manufactured,  but  a  great  many  of  these  will  be 
found  precisely  similar  in  their  colour  and  action, 
although  bearing  different  names,  while  others  are  at 
the  most  only  different  shades  of  the  same  colour. 
The  foregoing  list  gives  a  selection  of  the  most  useful 
of  these  dyes,  and  those  marked  with  an  asterisk  will 
be  found  to  give  the  best  results.  Many  of  the  others 
would,  however,  be  valuable  stains  if  any  means  could 
be  discovered  for  fixing  them  so  that  they  could  be 
passed  through  spirit  without  becoming  obliterated. 

8.  Soluble  Anilin  Blue  or  China  Blue.  This  is  a  use- 
ful stain  for  some  tissues,  such  as  stomach  and  spinal 
cord.  It  is  made  very  simply  by  taking  some  of  the 


On  Staining.  29 


granules,  and  placing  them  in  a  large  test-tube,  and 
adding  distilled  water  :  the  solution  ought  to  be  strong, 
nearly  saturated.  When  all  the  granules  have  dis- 
solved, filter  the  solution,  and  keep  it  in  a  stoppered 
bottle. 

Sections  that  are  to  be  passed  through  spirit  require 
to  be  rather  deeply  stained,  as  a  good  deal  of  the  colour- 
ing matter  will  come  out  in  the  spirit,  and  it  is  better 
to  remove  them  at  once  from  the  water  to  absolute 
alcohol,  as  the  colour  is  not  affected  so  much  by  it. 

9.  Pure  soluble  Anilin  Blue.     This  is  a  very  good  blue 
colour,  soluble  in  water :  it  is  more  brilliant  than  the 
former,  and  more  expensive  ;  the  China  blue  is,  in  fact, 
prepared  from  it.      A  saturated  solution  must  be  made 
and  great  care  used  in  staining  with  it,  as  its  action  is 
very  rapid.      Like  all  anilin  colours,  its  brilliancy  is 
impaired  by  a  long  immersion  in  spirit. 

This  colour  has  not  been  much  used  as  yet,  but  it 
will  probably  become  a  favourite  when  it  is  better 
known.  To  use  either  of  these  solutions,  a  few  drops 
are  placed  in  a  watch-glass,  and  diluted  with  water, 
one  or  two  drops  are  quite  sufficient,  the  sections  are 
then  washed  well  in  water. 

10.  Serge  Blue.     This  is  another  shade  of  blue  ;    a 
good  colour,  and  stains  well,  but  will  not  stand  immer- 
sion in  spirit. 

11.  Tyrian  Blue.     A  much  less  brilliant  colour  than 
any  of  the  others,  more  inclined  to  purple  :  it  does  not 
stand   spirit,    and   cannot   be   much   used  until   some 
method  is  found  for  fixing  it. 

12.  Safranine.     Is  a  good  deep   colour,  and   stains 


30  On  Staining. 


gland  tissue,  such  as  mucous  glands  in  the  tongue,  it 
does  not,  however,  stand  immersion  in  spirit  well,  and 
the  sections  require  to  be  deeply  stained.  It  has  been 
recommended  for  staining  amyloid  degeneration,  but 
does  not  excel  several  other  anilines  which  can  be  more 
easily  fixed. 

13.  Dahlia.     Bosanilin,   Methylanilin    Violet.      These 
three,  differing    but    little    in    colour,   may  be    con- 
sidered   together ;     they    can    be    partially  fixed    by 
passing    the    sections    through    a    1   per    cent,   solu- 
tion  of    hydrochloric   acid,  but   even    then   they  will 
not   stand  prolonged  immersion  in    spirit,   and  it  is 
better  to  remove  them  from  the  water  at  once  to  abso- 
lute alcohol.     The  solutions  must  be  rather  strong,  and 
the  sections  well  stained  before  they  are  removed  to  the 
acid  solution. 

14.  Malachite  Green.      A  very  beautiful  colour,  but 
will  not  stand  well :    it  is  almost  entirely  removed  by 
spirit,  and  is  therefore  not  of  much  use  to  the  Histolo- 
gist. 

15.  Iodine  Green.     A  darker  green  than  the  former, 
and  very  durable,  standing  spirit  well  and  not  fading, 
as  far  as  two  years  experience  goes.      It  is  an  invalu- 
able colouring  agent  in  double  staining,  as  it  is  not  so 
opaque  as  anilin  blue.      Make  a  saturated  solution  in 
water  and  filter,  place  a  few  drops  in  a  watch-glass 
and  dilute  :    it  is  very  strong,  and  the  section,  when 
taken  from  spirit,  will  float  on  the  surface,  when  it  may 
be  seen  taking  in  the  colour  :    if  a  light  stain  only  is 
required  it  will  be  sufficient  to  let  it  remain  on  the 
surface ;    but  if  a  darker  stain  is  wanted  it  must  be 


On  Staining.  31 


wholly  immersed,  and  then  it  must  not  be  left  long 
or  the  stain  will  be  too  deep,  and  it  cannot  be  after- 
wards removed. 

This  is  one  of  the  most  useful  of  the  anilins  soluble 
in  water,  and  the  results  when  it  is  carefully  used  are 
very  beautiful ;  it  picks  out  all  the  nuclei,  and  in  grow- 
ing bone  it  colours  the  unossified  cartilage,  giving  a 
very  striking  result.  It  is  also  a  most  valuable  anilin 
in  double  staining  ;  its  action  will  be  described  under 
that  head. 

16.  Bismarck  Brown.     This  is  a  good  colour,  a  deep 
rich  brown.     It  is  easily  soluble  in  spirit  but  does  not 
make  a  very  deeply  coloured  solution,  it  stains  well. 
An  aqueous  solution  can  be  made  by  the  addition  of  a 
little  dilute  acetic  acid,  this  solution  also  stains  well 
but  it  remains  to  be  seen  whether  it  will  fade  or  not. 
The  aqueous  solution  stains  gland  tissue  well,  and  in  a 
section  of  tongue  where  the  mucous  glands  are  deeply 
stained  with  the  aqueous  solution,  the  colour  has  not 
faded  after  an  exposure  of  two  months.     It  is  doubtful 
as  yet  which  will  prove  to  be  the  best  solution,  the 
aqueous  is  decidedly  the  richest  colour  to  the  eye  but 
must  be  kept  for  a  week,  after  making,  before  it  is 
used. 

ANILIN  DYES  SOLUBLE  IN  SPIRIT. 

17.  Spiller's  Purple.     This  is  a  very  good  colour  for 
use  in  double  staining,  but  it  is  very  difficult  to  fix  it 
and  if  the  section  is  passed  through  methylated  spirit, 
it  will  be  almost  all  washed  out ;  it  is  not  quite  so  easily 
acted  on  by  strong  absolute  alcohol,  '795  sp.  gr.,  but 


32  On  Staining. 


the  section  must  be  very  deeply  stained  to  get  enough 
left  in  it  to  make  a  satisfactory  specimen. 

There  are  several  other  colours  soluble  in  spirit  which 
are  much  easier  to  use  and  consequently  this  has  not 
been  much  tried,  but  it  would  well  repay  the  trouble 
if  some  means  were  found  to  make  it  more  permanent. 
A  saturated  solution  must  be  used,  and  it  is  better  not 
to  wash  the  section  too  much  in  spirit,  before  putting 
it  in  the  aqueous  solution  in  double  staining. 

18.  Rosein.     This  is  the  best  of  all  the  anilin  colours 
soluble  in  spirit  and  must  not  be  confounded  with  ros- 
anilin.      It  is   easily   soluble  in   spirit,   the   ordinary 
methylated  spirit  will  do,  its  action  is  rapid,  and  it  is 
better  to  use  a  strong  solution  and  immerse  the  section 
for  a  very  short  time. 

To  make  the  solution,  place  some  of  the  granules  in 
a  large  test  tube  and  nearly  fill  with  spirit,  cork  and 
shake  it  and  let  it  stand,  when  all  the  granules  are 
dissolved,  add  some  more  until  the  solution  appears 
to  be  saturated,  then  filter  and  keep  in  a  stoppered 
bottle.  Place  some  in  a  watch  glass  and  dilute  with 
about  an  equal  quantity  of  spirit ;  this  will  do  for  all 
ordinary  purposes. 

19.  Anilin  Eed.     This  is  another  shade  of  red  and 
does  not  differ  much  from  rosein  in  colour  while  it  has 
not  its  universal  applicability,  and  does  not  fix  so  well. 
It  has  not  been  much  used,  and  experiments  might 
bring  out  some  valuable  points  in  it  as  a  dye  for  special 
subjects.     It  is  prepared  and  used  in  the  same  manner 
as  rosein. 

20.  Anilin  Violet.     This  colour  comes  next  to  rosein 


On  Staining.  33 


in  utility  while  it  surpasses  it  in  beauty ;  it  is  not  quite 
so  easily  fixed,  otherwise  it  can  be  used  in  the  same 
manner  and  as  readily  as  rosein.  It  has  this  advan- 
tage over  rosein,  it  is  a  much  softer  colour  to  the  eye 
and  makes  a  really  beautiful  combination  with  some  of 
the  aqueous  solutions  in  double  staining. 

It  is  readily  soluble  in  spirit,  and  even  diluted  makes 
a  very  opaque  solution,  rendering  it  a  matter  of  diffi- 
culty to  find  the  sections  in  it ;  they  may  be  seen,  how- 
ever, by  holding  the  watch  glass  against  a  lamp  or 
bright  sky. 

Good  results  are  often  obtained  with  this  colour  by 
removing  sections  from  water  and  letting  them  spread 
out  a  little  on  the  needle  in  a  strong  solution  and 
then  removing  them  quickly  to  an  aqueous  solution  of 
another  color. 

21.  Citranine.     A  yellow  substance  insoluble  in  water 
and  only  soluble  in  spirit  by  the  application  of  heat. 
It  does  not  seem  to  give  a  deep  enough  colour  to  be  a 
useful  staining  agent  in  microscopical  investigations, 
but  a  more  extended  trial  may  bring  out  some  valuable 
peculiarities,  as  it  seems  to  differ  a  good  deal  from  the 
other  anilins. 

22.  Aurine.     This  is  another  colour  of  which  there 
is  not  much  known  of  its  action  on  animal  tissues  ;   as 
far  as  it  has  been  used,  it  has  not  given  any  satisfactory 
result. 

23.  Pure  Opal  Blue.     This  is  the  best  blue  soluble  in 
spirit,  and  is  therefore  useful  in  cases  where  an  aqueous 
stain  is  not  admissible.     It  is  made  by  dissolving  the 
powder   in   spirit   and  then   filtering  it.      Methylated 


34  On  Staining. 


spirit  will  do  if  strong,  but  spirit  of  wine  is  better.  A 
few  drops  are  taken  and  diluted  with  spirit,  and  the 
sections  immersed  from  three  to  five  minutes,  and  then 
washed  in  spirit. 

SELECTIVE  STAINS. 
These  will  be  found  under  their  respective  headings. 


CHAPTEE  V. 
DOUBLE  STAINING. 

BY  double  staining  is  meant  a  process  in  which  two 
colours  are  taken,  which  have  affinities  for  different 
elements  in  the  tissues  to  which  they  are  applied. 
Thus  while  one  colour  will  stain  the  connective  tissue 
and  protoplasm  of  cells,  the  other  will  colour  all  nuclei 
and  so  differentiate  the  different  elements  as  to  make 
them  more  easily  discernible.  Others  again  will  stain 
different  glands  according  to  their  secretions.  Thus 
showing  a  distinct  chemical  reaction  between  glands 
differing  in  their  functions. 

In  other  cases  the  duct  of  a  gland  can  be  stained  of  a 
different  colour  to  the  surrounding  tissue  and  its  own 
secreting  substance,  by  which  means  it  is  easy  to  distin- 
guish it,  and  thus  show  if  it  is  implicated  in  any  mor- 
bid change,  and  also  in  some  cases  prove  whether  the 
morbid  change  is  primary  in  it  or  has  extended  from 
surrounding  tissues,  in  which  case  all  the  ducts  would 
not  probably  be  similarly  affected. 

Double  staining  is  a  subject  that  requires  to  be  very 
much  more  worked  out  than  it  has  been  hitherto,  and 
in  the  present  work,  those  processes  only  will  be  given 
in  detail,  which  have  been  fairly  well  tried ;  many  other 
combinations  of  staining  agents  will  be  apparent  to  the 
student,  which  have  not  yet  been  worked  out,  for 
want  of  time,  and  there  is  no  pursuit  in  which  patience 

D2 


36  Picro-  carmine  and  Anilin. 

and  time  for  experimenting  are  more  required,  than  in 
double  staining. 

The  student  must  not  be  discouraged  by  many 
failures  as  there  is  always  some  new  fact  to  be  learnt 
and  noted,  and  in  the  application  of  these  to  future 
experiments,  some  brilliant  results  are  sure  to  be  ob- 
tained. 

PlCKO-CAKMINE    AND    LOGWOOD. 

This  combination  has  been  used  for  a  long  time,  and 
gives  very  satisfactory  results.  The  student  should 
begin  with  sections  of  scalp,  skin,  or  tongue,  and  the 
result,  if  the  process  be  carefully  carried  out,  will  be 
found  very  satisfactory. 

The  sections  must  be  first  stained  in  picro- carmine 
and  then  in  logwood.  Make  a  dilute  solution  of  picro- 
carmine  in  distilled  water,  about  10  drops  to  the  watch 
glass,  and  let  the  sections  remain  in  it  for  from  twenty 
minutes  to  half  an  hour,  then  wash  in  water  and  place 
in  distilled  water  acidulated  with  1  or  2  drops  of  acetic 
or  picric  acid.  Let  them  remain  in  this  for  about  an 
hour.  Eemove  the  sections  from  the  acidulated  water 
and  place  them  in  dilute  logwood  stain  ;  this  should  not 
be  too  strong,  from  5  to  7  drops  to  the  watch  glass  of 
distilled  water;  do  not  let  them  stain  too  deeply. 
When  sufficiently  coloured,  which  will  be  shown  by 
their  becoming  a  faint  lilac  colour,  they  must  be 
washed  to  remove  the  excess  of  logwood,  and  mounted 
in  the  usual  manner. 

This  double  stain  is  very  effective  when  used  with 
fresh  tissues,  such  as  serous  membranes  ;  it  brings  out 


Staining  Process.  37 

the  connective  tissue  corpuscle  in  the  mesentery  of  the 
newt,  and  at  the  same  time  shows  the  non-striped 
muscle  tissue  very  well.  It  is  also  useful  in  bringing 
out  the  delicate  tissue  in  the  tubuli  seminiferi  of  the 
testis  and  showing  the  developing  spermatozoa  there. 

In  any  tissue  when  there  are  elements  of  different 
kinds,  such  as  scalp  or  developing  bone,  it  will  be 
found  to  give  very  good  results.  The  logwood  stain 
must  not  be  too  deep,  as  it  is  a  very  opaque  colour. 

CARMINE  AND  INDIGO- CARMINE. 

This  is  a  very  useful  double  stain,  and  is  especially 
applicable  to  sections  made  from  material  hardened  in 
chromic  acid  mixture  as  they  do  not  require  to  be 
passed  through  a  solution  of  bicarbonate  of  soda,  but 
can  be  placed  at  once  in  the  carmine  solution. 

In  this  staining  process  three  solutions  are  necessary. 

I.  Carmine  solution  (page  24). 

II.  Hydrochloric  acid,  1  part ;    absolute  alcohol  '795, 
9  parts. 

III.  Indigo  carmine  solution  (page  25). 

STAINING  PROCESS. 

Take  a  few  drops  of  No.  1  in  a  watch  glass  and  im- 
merse the  sections,  let  them  remain  for  two  or  three 
minutes  and  then  remove  them  to  a  watch  glass  con- 
taining a  small  quantity  of  No.  2.  Let  them  remain 
in  this  until  they  take  on  a  bright  rose  colour,  which 
will  be  in  a  few  seconds,  then  wash  them  in  methyl- 
ated spirit  to  get  rid  of  the  acid.  They  must  be  washed 
in  several  changes  of  spirit.  When  the  acid  has  been 


38  Pier o- carmine  and  Anilin  Colours. 

thoroughly  removed,  place  the  sections  in  a  little  of  No. 
3  solution  undiluted,  and  let  them  remain  in  it  until 
they  show  a  distinct  blue  tinge :  the  proper  depth  of 
this  staining  will  be  learnt  by  practice. 

When  carefully  used  this  staining  process  is  an  ad- 
mirable one,  but  there  are  one  or  two  points  that 
have  to  be  attended  to,  or  the  two  colours  will  not 
be  sufficiently  differentiated.  If  the  section  is  left 
too  long  in  the  acid  mixture  the  carmine  will  be 
taken  out  of  the  edges  and  these  parts  will  after- 
wards take  on  the  blue  stain  deeply,  and  so  give  a 
result  the  very  opposite  of  that  intended,  as  the 
whole  value  of  double  staining  depends  on  one  co- 
lour picking  out  the  whole  of  one  particular  tissue 
throughout  the  section,  and  if  this  is  not  done  the  spe- 
cimen is  of  no  use.  If  the  carmine  stain  is  only  just 
sufficiently  acted  on  by  the  acid,  so  as  to  change  the 
original  dull  purple  colour  to  a  bright  rose,  and  the 
edges  of  the  specimen  are  not  bleached,  it  will,  when 
put  in  the  indigo -carmine  solution,  stain  evenly  through- 
out. 

If  the  acid  solution  be  too  strong  it  will  have  the 
same  effect  as  too  long  immersion  in  a  weaker  solu- 
tion, and  a  few  seconds  will  bleach  the  edges.  This 
process  will  be  found  very  useful  in  pathological  inves- 
tigation, as  the  carmine  picks  out  very  distinctly  all  the 
new  growths. 

PlCRO-CAKMINE    AND    ANILIN    COLOUES. 

Some  very  good  results  may  be  obtained  by  staining 
sections  first  in  pier  o- carmine,  then  letting  them  remain 


Treble  Staining.  39 


in  acidulated  water  for  an  hour,  and  afterwards  stain- 
ing them  with  various  solutions  of  anilin  colours. 

Safranine,  after  picro-carmine,  gives  a  good  double 
stain,  as  the  picro-carmine  colours  all  the  connective 
tissue  and  nuclei,  while  the  safranine  stains  muscle, 
epithelium,  &c. ;  but  the  two  colours  are  not  sufficiently 
different  to  give  as  good  a  result  as  logwood  and  picro- 
carmine,  although  they  will  be  found  useful  where  great 
transparency  is  desired. 

Picro-carmine  and  iodine  green  give  a  very  beautiful 
effect  when  it  is  wanted  to  isolate  gland  tissue ;  such 
as  Peyer's  patches,  or  the  glands  in  the  tongue,  oeso- 
phagus, or  solitary  glands  in  the  large  intestine.  The 
picro-carmine  staining  everything  but  the  glands,  which 
remain  a  bright  green. 

Eosin  and  anilin  blue  give  good  results,  but  require 
to  be  used  cautiously,  as  if  the  staining  is  too  deep  the 
section  becomes  opaque.  To  get  the  best  effect,  the 
section  should  be  very  thin,  and  must  be  well  washed 
after  staining  with  eosin,  and  then  just  immersed  for  a 
few  seconds  in  the  anilin  blue. 

A  great  many  other  combinations  will  suggest  them- 
selves to  the  student,  and  he  will  be  amply  rewarded  by 
experimenting  further  with  the  various  staining  agents 

mentioned. 

TREBLE  STAINING. 

The  combination  which  has  given  the  best  effect  so 
far  in  treble  staining  is :  picro-carmine,  rosein,  and 
iodine  green.  Stain  the  sections  well  according  to  the 
process  already  described  for  picro-carmine,  and  soak 
them  in  acidulated  water.  Then  take  a  few  drops  of 


40  Treble  Staining. 


rosein  solution,  No.  18  dilute  it  with  spirit,  and  im- 
merse the  sections  for  two  or  three  minutes,  remove 
them  to  methylated  spirit  and  wash  off  the  excess  of 
colouring  matter.  Then  place  them  in  a  dilute  solution 
of  iodine  green.  Coming  from  spirit  they  will  float  on 
the  top  of  the  watery  solution,  and  this  in  many  cases, 
when  the  green  stain  is  not  required  to  be  very  deep,  is 
quite  sufficient.  When  a  deeper  stain  is  required,  im- 
merse them  altogether,  and  let  them  remain  a  minute 
or  two  ;  but  it  must  be  borne  in  mind  that  this  colour 
cannot  be  washed  out  again  if  too  deep,  which  the 
spirituous  stain  can,  so  that  it  is  better  to  have  a  sec- 
tion apparently  over-stained  in  the  rosein  solution, 
while  it  is  even  under- stained  in  the  iodine  green. 
After  washing,  the  sections  are  mounted  in  the  usual 
manner.  It  will  be  found,  however,  that  a  good  deal 
of  the  rosein  will  come  out  in  the  second  immersion 
in  spirit,  and  it  is  necessary  to  change  it  until  no 
more  colour  comes  away ;  otherwise  the  oil  of  cloves 
will  become  coloured,  and  from  it  the  Canada  balsam, 
in  which  the  specimen  is  mounted. 

With  the  above  mentioned  three  colours,  the  most 
beautiful  effect  may  be  obtained,  but  it  will  take  some 
time  and  practice  to  get  the  process  exactly  right,  and 
this  is  a  matter  which  can  only  be  gained  by  experi- 
ence. The  results  will  be  found  to  vary  with  the 
length  of  time  the  section  is  immersed  in  each  of  the 
two  last  colours,  and  also  with  the  strength  of  the 
solutions. 

The  section  should  be  uniformly  and  deeply  stained 
with  picro-carmine.  The  other  two  solutions  should  be 


Treble  Staining.  41 


saturated  in  the  first  instance,  and  then  diluted  one- 
half  at  least.  If  they  are  to  be  laid  aside  for  some  time 
before  mounting  they  should  not  be  left  in  spirit,  but  in 
oil  of  cloves.  Only  a  few  sections  should  be  stained  at 
one  time  or  some  will  be  found  much  more  deeply 
stained  than  others.  The  best  results  will  also  be  ob- 
tained with  material  that  has  been  hardened  in  chromic 
acid. 

This  staining  process  is  well  shown  in  a  section  of 
the  base  of  a  cat  or  dog's  tongue,  cut  through  one  of 
the  circumvallate  papilla?,  the  section  should  be  suffi- 
ciently large  to  include  some  of  the  mucous  glands,  of 
which  there  are  a  large  number  in  that  region. 

If  the  staining  is  well  done  it  will  show  all  the 
muscle  fibres  stained  with  pier o- carmine,  the  connec- 
tive tissue,  protoplasm  of  cells,  &c.,  stained  with  rosein  ; 
while  all  the  nuclei  in  the  superficial  epithelium,  serous 
glands,  non-striped  muscle  tissue  in  the  vessels,  and 
elsewhere,  are  stained  a  brilliant  green. 

The  most  important  fact  demonstrated  by  this  pro- 
cess is  the  different  chemical  reaction  shown  by  the 
various  glands.  In  the  mucous  glands,  while  the  epi- 
thelium lining  the  duct  is  stained  in  precisely  the  same 
manner  as  the  superficial  epithelium  of  the  organ,  it 
will  be  found  that  the  moment  the  secreting  epithelium 
is  reached  a  new  colour  presents  itself,  which  differs  in 
toto  from  either  of  those  employed  in  the  process  ;  thus 
showing  that  the  secretion  has  the  power  of  causing 
these  two  colours,  green  and  red,  to  combine,  forming 
different  shades  from  purple  to  blue,*  according  to 

*  Iodine  green  is  a  very  blue  green. 


42  Chloride  of  Gold  and  Anilines. 

which  colour  predominates.  In  the  serous  glands,  how- 
ever, quite  another  aspect  is  presented;  there  is  no 
combination  as  in  the  mucous  glands,  but  the  proto- 
plasm of  the  cells  is  stained  more  or  less  deeply  with 
rosein,  while  the  nuclei  have  taken  on  the  green  ;  the 
colour  differs,  however,  from  that  of  the  surface  epithe- 
lium, and  appears  to  have  taken  on  picro-carmine  to 
some  extent,  which,  with  the  rosein,  gives  a  dull  red 
colour. 

In  many  places  will  also  be  seen  small  masses  of 
adenoid  tissue  which  have  stained  a  bright  green 
throughout. 

Altogether  this  makes  one  of  the  most  brilliant  speci- 
mens in  the  whole  range  of  histology,  and  although  the 
process  is  rather  troublesome,  and  requires  a  certain 
amount  of  practice  to  determine  the  time  required  for 
each  immersion,  it  amply  repays  when  once  properly 
done. 

Take  only  a  few  sections  at  a  time,  and  do  not  hurry 
over  the  different  processes,  and  after  a  few  trials  the 
exact  time  of  immersion  will  be  hit  on,  and  should  be 
recorded. 

CHLORIDE  OF  GOLD  AND  ANILINES. 

Some  very  striking  results  may  be  obtained  by  first 
staining  fresh  tissues,  especially  growing  bone,  in  chlo- 
ride of  gold  solution  (page  7),  and  then  decalcifying 
and  hardening  in  spirit.  After  the  material  has  har- 
dened sufficiently,  sections  may  be  made  and  stained 
with  two  colours.  It  is  not  quite  clear  what  action  the 
gold  chloride  has  on  those  parts  it  does  not  stain,  but 


Chloride  of  Gold  and  Anilines.  43 

that  it  has  some,  is  evident  from  the  difference  of  the 
action  of  anilin  dyes  on  those  specimens  prepared  in 
gold,  from  those  hardened  in  any  other  manner. 

A  very  good  material  for  the  purpose  is  the  tail  of  a 
young  rat  or  mouse,  placed  in  half  per  cent,  solution  of 
gold  chloride  for  an  hour  or  two,  and  then  decalcified 
and  hardened  in  the  usual  way.  Very  thin  transverse 
sections  should  be  cut,  and  stained  first  in  rosein  and 
then  in  iodine  green. 

On  examining  the  specimen  the  gold  staining  will  he 
seen  on  the  periphery,  bringing  out  the  tendon  cells, 
and  giving  a  dark  hue  to  everything  for  a  certain  dis- 
tance from  the  outside  ;  but  within  this  a  great  variety 
of  colour  will  be  found,  the  different  tissues  being 
stained  in  a  most  gorgeous  manner.  In  the  middle 
the  bone  trabeculse  will  be  seen  faintly  stained,  while 
the  calcified  cartilage,  in  their  centres,  is  stained  a 
bright  colour,  totally  different.  All  these  colours  may 
be  varied  by  using  different  anilin  solutions,  and  a  very 
pretty  result  may  be  obtained  by  simply  staining  with 
iodine  green.  In  the  above  instance  the  true  bone  is 
only  faintly  stained,  while  the  calcified  cartilage  takes 
the  colour  deeply  ;  this  may  be  reversed  by  using  the 
carmine  and  indigo -car  mine  process  after  the  gold,  when 
the  bone  will  be  found  deeply  stained,  while  the  calci- 
fied cartilage  is  not  stained  at  all,  and  looks  like  a  clear 
space  in  the  bone  trabeculas.  These  two  processes  will 
be  found  invaluable  in  any  investigation  into  the  deve- 
lopment of  bone. 


CHAPTEE  VI. 
ON  MOUNTING. 

SLIDES. 

Glass  slides,  3x1,  must  be  cleaned  before  using, 
and  a  good  plan  is  to  keep  some  cleaned,  ready  for  use, 
in  a  two-dozen  box  with  rack  work,  where  they  stand 
on  their  edges  and  do  not  get  dusty.  The  ordinary 
slides  sold  at  the  shops  at  6s.  a  gross,  are  easily  cleaned 
with  a  chamois  leather.  Sometimes,  however,  especially 
when  using  slides  for  the  second  time,  they  cannot  be 
cleaned  so  readily,  and  they  must  be  soaked  in  a  decoc- 
tion of  oak  galls  for  some  hours  ;  this  is  made  by  pour- 
ing boiling  water  on  bruised  oak  galls  and  straining. 

COVER  GLASSES. 

The  usual  size  of  these  is  f  of  an  inch  square,  but 
larger  ones  will  be  required,  and  some  of  |  of  an  inch 
should  be  obtained.  For  practical  men  square  cover 
glasses  are  the  best ;  but  where  aesthetic  mounting  is 
the  order  of  the  day,  round  cover  glasses  and  variously 
coloured  cements  for  sealing,  with  the  help  of  a  turn- 
table will  be  required ;  these,  for  obvious  reasons,  will 
not  be  considered  in  this  work. 

Thin  glass  is  made  by  Messrs.  Chance,  of  Birming- 
ham, in  three  thicknesses,  designated  by  the  numbers 
1,  2,  and  3  ;  of  these  No.  1  is  the  thinnest.  This  is  the 
best  to  use.  It  varies  in  thickness  from  '004,  or  even 
thinner  to  -008. 


On  Mounting.  45 


ON  MEASURING  COVEK  GLASSES. 

For  ordinary  students'  work  the  No.  1  cover  glasses 
will  do  perfectly  well,  as  they  are  very  near  the  thick- 
ness to  which  object  glasses,  without  a  correction  collar, 
are  adjusted ;  but  when  high  power  glasses  are  to  be 
used,  it  facilitates  the  work  very  much,  to  know  the 
thickness  of  the  cover  glass  under  which  the  specimen 
is  mounted,  and  with  very  high  powers,  or  those  with 
wide  angle  of  aperture,  the  cover  must  be  at  least  '004 
to  enable  the  glass  to  work  through  it.  Powell  and 
Lealand's  -^  water  immersion  requires  a  cover  glass 
•003  of  an  inch. 

To  save  expense,  as  dealers  charge  a  high  price  for 
very  thin  measured  glass,  the  student  will  find  it  a 
great  advantage  to  purchase  a  small  American  steel 
gauge,  sold  by  Buck,  Holborn  ;  it  is  made  for  measur- 
ing the  thickness  of  sheet  brass  in  fine  work,  but  does 
admirably  for  measuring  cover  glasses  ;  the  way  to  use 
it  will  be  at  once  apparent.  With  this  instrument  let 
the  student  measure  an  ounce  of  No.  1  glass,  and  select 
all  those  that  are  '004  and  under,  they  may  be  put 
away  for  future  use.  This  is  not  much  trouble,  and  a 
few  of  these  very  thin  glasses  will  last  a  long  time,  as  it 
is  only  in  special  work  that  they  will  be  required.  The 
oil  immersion  lenses  have  also  done  away  in  a  great 
measure  with  the  necessity  of  using  measured  cover 
glasses. 

ON  CLEANING  COVEE  GLASSES. 

The  following  plan  will  be  found  a  very  good  one, 
both  in  the  saving  of  time  and  breakage. 


46  On  Mounting. 


Place  the  cover  glasses  to  be  cleaned  in  a  glass 
vessel  containing  strong  sulphuric  acid,  and  agitate 
gently  until  the  acid  has  penetrated  between  the 
glasses  and  driven  out  the  air-bubbles,  let  them  remain 
in  this  for  an  hour  or  two  and  then  wash  well  in  water 
until  no  acid  is  left.  Eemove  them  to  a  capsule  con- 
taining methylated  spirit.  Take  out  each  one  separ- 
ately with  a  pair  of  broad  pointed  forceps,  and  wipe  dry 
with  a  silk  or  soft  linen  rag. 

With  very  thin  cover  glasses,  such  as  -003,  each 
glass  may  be  dipped  in  absolute  alcohol  when  taken  out 
of  the  methylated  spirit  and  then  carefully  dried  with 
an  old  silk  handkerchief. 

MOUNTING  FLUIDS. 

For  fresh  tissues : 

Glycerine . 
For  hardened  tissues  : 

Canada  balsam  solution. 
Dammar  varnish. 

MOUNTING  FKESH  TISSUES, 

Place  the  tissue  to  be  mounted  in  a  capsule  of  water 
of  sufficient  depth  to  cover  more  than  half  of  an  ordi- 
nary glass  slide,  when  placed  in  it  with  one  end  on  the 
bottom  and  the  other  resting  on  the  opposite  side. 
With  a  needle,  bring  the  tissue  over  the  middle  of  the 
slide  and  hold  it  there,  at  the  same  time  raise  the  upper 
end  of  the  slide  very  gently,  so  that  the  tissue  will 
adhere  to  it  and  be  raised  out  of  the  water.  See  that  it 


On  Mounting.  47 


is  not  folded  in  any  part.  Lay  the  slide  on  some  filter 
paper,  and  with  needles  spread  out  the  tissue  to  its 
fullest  extent,  without  stretching  it.  It  is  necessary  to 
be  very  careful  of  this,  as  if  the  tissue  be  a  serous  mem- 
brane, stained  with  silver,  the  outlines  of  the  cells  will 
be  completely  destroyed  wherever  it  has  been  stretched. 
In  the  same  way,  non- striped  muscle  fibre  in  the  me- 
sentery of  the  newt,  will  be  broken  up  and  quite  ruined. 

When  the  tissue  appears  to  be  extended  in  a  natural 
manner,  without  folds,  take  up  the  slide  and  wipe  off 
all  moisture  from  it  with  a  clean  cloth,  if  there  is  a 
large  quantity  on  the  specimen,  some  may  be  removed 
with  a  bit  of  filter  paper,  but  great  care  must  be  taken 
not  to  touch  the  specimen  itself  with  the  paper  as  it 
will  adhere  to  it ;  at  the  same  time  it  must  not  be 
allowed  to  become  dry,  and  if  this  seems  probable,  it 
can  easily  be  moistened  by  breathing  on  it  occasionally, 
until  the  cover  glass  is  ready.  Take  up  a  clean  cover 
glass  and  place  a  drop  of  glycerine  on  the  centre,  invert 
and  place  it  horizontally  on  the  specimen,  leaving  the 
weight  of  the  cover  glass  to  spread  out  the  glycerine. 
If  there  is  an  excess  of  glycerine  round  the  edges  of  the 
cover  glass,  it  must  be  removed  by  placing  small  pieces 
of  filter  paper  in  contact,  which  will  soon  absorb  the 
superfluous  fluid,  but  must  not  be  left  too  long  or  they 
will  drain  it  from  under  the  cover  glass.  When  suffi- 
cient has  been  taken  up  by  the  pieces  of  filter  paper, 
remove  them  and  wipe  the  slide  with  a  dry  cloth,  tak- 
ing care  to  clean  off  all  glycerine  without  touching  the 
cover  glass. 

When  this  is  done  the  preparation  must  be  sealed,  by 


48  On  Mounting. 


painting  round  the  cover  glass  with  either  Dammar 
varnish  or  Hollis'  glue,  taking  care  that  only  the  ex- 
treme edge  of  the  cover  glass  is  included.  It  will  be 
necessary  to  give  a  second  and  third  coat  if  Dammar 
varnish  is  used,  at  intervals  of  a  few  days. 

It  will  be  found  a  good  plan  to  seal  first  with  Dam- 
mar varnish,  and  afterwards  to  cover  this  with  Hollis' 
glue,  as  it  makes  the  preparation  more  secure,  and  it  is 
absolutely  necessary  to  have  them  sealed  with  Hollis' 
glue  when  oil  immersion  lenses  are  to  be  used,  as  the 
cedar  oil  does  not  touch  it,  while  it  dissolves  Dammar 
varnish  at  once. 

Specimens  carefully  prepared  in  the  above  manner 
may  be  kept  for  years  without  deteriorating. 

MOUNTING  IN  CANADA  BALSAM  OR  DAMMAR. 
Canada  balsam  mounting  fluid  is  prepared  by  mixing  : 
Canada  balsam,  105  parts. 
Turpentine,  35  parts. 
Chloroform,  35  parts. 

Dammar  varnish  is  prepared  thus  : 
Take  of 

Gum  Dammar  in  powder,  i  oz.,  and  dissolve  it  in 

turpentine,  1-J-  oz.     Filter. 
Gum  mastic,  i  oz.,  and  dissolve  it  in  chloroform, 

2  oz.     Filter. 
Mix  the  two  solutions  and  filter  again. 

Put  in  stoppered  bottles,  and  see  that  they  are  per- 
fectly free  from  moisture  before  using.  A  small  drop- 
bottle  of  each  of  these  fluids  must  be  kept  for  daily 


On  Mounting.  49 


use,  and  when  they  get  thick  from  evaporation  a  little 
chloroform  can  be  added. 

Both  of  these  mounting  fluids  are  used  in  the  same 
manner  and  one  description  will  apply  equally  well  to 
each.  Canada  balsam  is  the  one  commonly  used,  as 
the  materials  of  which  it  is  composed  are  very  cheap, 
while  gum  dammar  is  rather  expensive.  The  Dammar 
varnish  is  also  sometimes  apt  to  become  cloudy  after  a 
time  and  it  is  difficult  to  make. 

To  MOUNT  IN  CANADA  BALSAM  OK  DAMMAR  YARNISH. 

The  sections  having  been  properly  stained  and 
washed,  are  placed  in  methylated  spirit  to  remove 
some  of  the  water,  and  then  immediately  transferred 
to  a  small  quantity  of  absolute  alcohol  in  a  watch 
glass,  and  covered  with  another  to  prevent  evaporation. 
They  should  be  left  in  this  for  about  10  minutes. 
The  absolute  alcohol,  which  should  be  the  strongest 
sp.  gr.  -795,  has  a  great  affinity  for  water,  and  will 
remove  all  that  is  in  the  sections. 

"When  ready  remove  the  sections  one  by  one  from  the 
absolute  alcohol  with  a  needle,  and  drain  off  as  much 
alcohol  as  possible  by  touching  the  section  on  the  back 
of  the  hand  or  on  a  piece  of  clean  filter  paper,  the  back 
of  the  hand  is  the  best,  as  some  fibres  from  the  filter 
paper  may  adhere  to  the  section,  which  when  seen 
under  the  microscope,  will  not  improve  the  beauty  of 
the  preparation;  when  sufficiently  drained,  without 
being  allowed  to  become  absolutely  dry,  they  are  placed 
in  a  vessel  containing  oil  of  cloves  ;  they  will  spread 
out  on  the  surface  of  the  oil,  and  as  the  spirit  evapo- 

E 


50  On  Mounting. 


rates  they  will  become  completely  permeated  with  it 
and  very  transparent.  If  there  are  any  folds  these 
should  now  be  straightened  out  carefully  with  needles. 

Having  placed  a  drop  of  Canada  balsam  on  the  slide, 
spread  it  out  slightly  with  a  needle,  select  a  section  in 
the  oil  of  cloves,  and  pass  the  copper  lifter  under  it, 
raise  the  lifter  and  hold  the  section  in  position  with  a 
needle  by  its  upper  corner,  and  having  made  sure  there 
are  no  folds,  remove  the  lifter  with  the  section  on  it 
from  the  oil  of  cloves,  let  as  much  oil  drain  off  as  possi- 
ble against  the  side  of  the  vessel,  and  remove  the  rest 
by  placing  the  edge  of  the  lifter  on  a  piece  of  filter 
paper.  Place  the  edge  of  the  lifter  on  the  slide  in  the 
drop  of  Canada  balsam,  and  gently  draw  down  the 
section  with  a  needle  as  soon  as  a  corner  projects  from 
the  lifter  on  to  the  slide,  hold  it  there  lightly  with  the 
needle  and  slowly  draw  away  the  lifter  ;  if  this  is  care- 
fully done  the  section  will  lie  in  its  place  in  the  middle 
of  the  slide  without  any  folds. 

A  lifter  is  made  by  beating  out  the  end  of  a  copper 
wire,  filing  it  smooth,  and  then  turning  up  the  broad 
portion  slightly. 

Take  up  a  cover  glass  with  the  broad  pointed  forceps 
and  hold  it  between  the  thumb  and  fore-finger  of  the 
left  hand,  place  a  small  drop  of  Canada  balsam  on  its 
lower  edge,  transfer  to  the  right  hand  and  gently  lower 
it  on  to  the  section,  keeping  the  left  thumb  against  one 
corner  to  prevent  its  slipping,  and  gradually  lower  it  by 
removing  the  right  hand  very  slowly,  watching  all  the 
time  to  see  that  no  air  bubble  is  entangled  in  the 
section. 


On  Mounting.  51 


With  a  little  practice  this  can  be  done  very  neatly 
without  an  air  bubble  in  any  part  of  the  preparation ;  it 
requires  patience,  however,  and  it  is  no  use  to  try  air 
pumps  or  any  dodges,  to  remove  the  bubbles,  as  they 
are  useless  ;  the  only  thing  to  be  done  when  an  air 
bubble  lodges  in  a  cavity  of  the  section  and  refuses  to 
move  in  any  way  by  gentle  pressure,  is  to  lift  the  cover 
glass,  and  transfer  the  section  to  oil  of  cloves,  and  then 
remount  it. 

When  several  sections  are  to  be  mounted  on  one 
slide,  a  slight  pressure  on  each  with  the  needle  will 
generally  retain  it  in  its  position,  if  too  much  of  the 
mounting  fluid  is  not  used. 

It  will  often  be  found  on  examining  preparations 
after  they  have  been  mounted  some  little  time,  that 
the  fluid  has  evaporated  and  left  a  vacuum  under  the 
cover  glass ;  in  this  case  a  drop  of  the  mounting 
fluid  must  be  placed  on  the  slide  in  contact  with  the 
cover  glass,  and  it  will  immediately  run  in  and  fill  up 
the  empty  space,  provided  always  an  egress  has  been 
allowed  to  remain  for  the  contained  air,  when  this  is 
impossible  from  the  small  size  of  the  hole  at  the  edge 
of  the  cover  glass,  the  only  thing  to  be  done  is  to  wait 
until  some  of  the  material,  of  which  the  mounting  fluid 
is  composed,  has  been  dissolved  by  the  fresh  fluid. 
Applying  heat  will  effect  it,  and  at  the  same  time  in 
all  probability  ruin  the  specimen. 

Each  preparation  should  be  examined  under  the  mi- 
croscope and  if  found  to  be  worth  keeping,  labelled. 
On  the  label  should  be  noted  the  tissue,  date  of  its  pre- 
paration, mode  of  hardening  and  staining,  thickness  of 

E2 


52  On  Mounting. 


cover  glass  if  it  has  been  measured,  and  anything  of 
note  which  may  be  seen  at  the  time  it  is  examined. 
Exceptionally  good  sections  should  always  have  a  pri- 
vate mark  to  show  that  they  are  not  to  be  given  away 
or  exchanged. 

They  should  be  kept  in  a  cabinet  where  they  may  lie 
flat. 

ON  BKEAKING  DOWN  OLD  PKEPAEATIONS. 

It  is  often  necessary  to  break  down  an  old  prepara- 
tion and  remount  it.  The  cover  glass  may  be  broken, 
the  staining  faded,  or  the  cover  glass  may  be  too  thick, 
and  preparations  should  never  be  discarded  for  these 
reasons,  as  it  is  quite  easy  to  remount  them.  When  a 
specimen  has  been  mounted  in  glycerine,  it  is  an  easy 
matter  to  remove  the  cover  glass,  all  that  is  necessary 
being  to  cut  round  the  cement  with  a  sharp  knife,  lift 
the  cover  glass  carefully  with  a  needle,  and  float  off 
the  section  in  water ;  if  it  is  very  delicate  the  cover 
glass  had  better  be  removed  under  water.  The  section 
can  then  be  washed,  to  remove  the  glycerine,  and  re- 
stained  if  required ;  it  will  then  be  ready  for  mounting 
in  the  usual  manner. 

To  break  down  a  specimen  mounted  in  Canada  bal- 
sam or  Dammar  varnish  is  more  difficult,  especially  if 
it  has  been  mounted  long  enough  to  allow  the  balsam 
or  Dammar  to  become  hard.  It  must  be  placed  in  a 
bath  of  chloroform  until  it  becomes  soft  enough  to  re- 
move the  cover  glass,  and  this  may  be  facilitated  by 
passing  the  slide  over  the  flame  of  a  spirit  lamp  so  as 
to  heat  it  very  slightly,  but  this  requires  care  as  the 
section  may  be  easily  ruined. 


On  Mounting.  53 


After  the  cover  glass  has  been  removed  the  section 
must  be  floated  off  into  chloroform  until  all  the  balsam 
or  Dammar  has  been  dissolved  out  of  it,  and  then  placed 
in  alcohol  for  a  short  time  ;  it  may  then  be  re- stained  if 
necessary  and  mounted  again. 

MOUNTING  LAKGE  SECTIONS. 

In  manipulating  large  sections  it  is  rather  difficult  to 
pass  them  through  the  different  processes  without  in- 
jury. This  may  generally  be  done  with  care,  and  they 
may  even  be  double  stained. 

There  are  some  tissues,  however,  so  fragile  that  they 
cannot  be  lifted  on  the  needle  without  tearing,  and 
these  must  be  left  in  one  vessel  and  the  different  pro- 
cesses applied  to  them  there.  This  is  not  a  very  satis- 
factory method,  as  the  staining  cannot  be  so  well  done 
unless  a  large  quantity  of  fluid  is  used,  and  every  section 
carefully  separated  from  its  neighbour,  they  are  also  apt 
to  be  injured  in  pouring  off  the  different  fluids.  This  is 
only  required  in  exceptional  cases.  It  is  when  the  mount- 
ing from  the  oil  of  cloves  comes,  that  the  difficulty  is 
experienced ;  as,  even  if  a  lifter  is  specially  made  large 
enough  to  take  up  a  whole  section,  the  adhesion  of  the 
section  to  such  a  large  surface  is  so  great,  that  it  is  im- 
possible to  get  it  off  without  tearing,  if  the  section  is  as 
thin  as  it  ought  to  be.  It  may,  however,  be  done  by 
using  the  cover  glass  as  a  lifter  in  the  following  man- 
ner. 

Take  as  an  example  a  longitudinal  section  of  kidney 
of  large  dog  or  man,  having  been  safely  stained,  it  lies 
in  the  oil  of  cloves  ready  to  be  transferred  to  the  slide. 


54  On  Mounting. 


The  section  measures  say  about  1J  inches  by  1  inch, 
some  slides  must  be  procured  3  by  2  inches  and  some 
cover  glasses  2  by  1£  inches,  these  had  better  be  of  No. 
3  thin  glass. 

Having  cleaned  one  of  the  slides,  place  some  Canada 
balsam  on  it  and  spread  it  out  with  the  needle,  to  some- 
thing near  the  size  of  the  specimen,  then  take  the  cover 
glass  and  pass  it  into  the  oil  of  cloves  under  the  speci- 
men, in  the  same  way  the  copper  lifter  is  used  to  smaller 
sections  ;  lift  the  cover  glass  and  keep  the  section  in  its 
place,  then  drain  off  the  superfluous  oil  by  holding  the 
cover  glass  on  filter  paper,  on  lifting  it  fresh  from  the 
oil  it  should  be  allowed  to  drain  slowly  from  one  corner, 
then  invert  the  cover  glass  with  the  section  on  it,  place 
a  little  Canada  balsam  at  the  lower  edge,  and  lower  it 
gently  into  the  Canada  balsam  on  the  slide  ;  this  must 
be  done  very  carefully,  as  bubbles  will  be  found  here 
and  there,  and  the  cover  must  be  lifted  a  little  and 
lowered  again,  until  they  have  all  been  driven  out.  It 
is  a  tedious  process  but  amply  repays  the  trouble. 

The  great  drawback  in  this  method  is  that  the  front 
of  the  cover  glass  is  covered  with  oil  of  cloves  and  can- 
not be  cleaned  until  the  balsam  sets,  a  matter  of  time 
with  such  a  large  surface.  It  can  certainly  be  sealed 
up  with  Hollis'  glue,  but  even  then  it  is  not  safe  and 
requires  a  great  many  coats  before  the  glue  is  suffi- 
ciently strong  to  resist  such  pressure  as  is  required  to 
clean  the  cover  glass. 

With  some  tissues  it  is  possible  to  use  a  large  cover 
glass  as  a  lifter,  and  by  allowing  a  large  quantity  of  oil 
of  cloves  to  remain  to  draw  it  off  on  to  the  slide  and  cover 


On  Mounting.  55 


in  the  usual  way,  but  with  other  tissues  such  as  testis 
this  is  utterly  impossible  if  the  sections  are  thin,  and 
they  can  only  be  mounted  in  the  manner  first  men- 
tioned. 

THIN  SLIDES. 

For  preparations  to  be  examined,  under  very  high 
powers,  thin  slides  made  from  glass  called  9  or  10  oz. 
crown  are  useful,  as  they  allow  the  condenser  to  come 
close  to  the  object. 


CHAPTEE  VII. 
METHOD  OF  OBTAINING  ANIMAL  TISSUES  FOE  EXAMINATION. 

THE  animals  required  will  be  a  cat,  rabbit,  and  guinea 
pig ;  and  for  some  special  tissues,  a  frog,  salamander, 
and  newt. 

To  kill  the  first  three  animals,  place  them  in  .a 
box  with  a  tight- fitting  lid,  having  previously  intro- 
duced a  sponge  saturated  with  chloroform.  In  this 
confined  space  the  chloroform  will  render  the  animals 
insensible  in  a  very  short  time,  and  then  kill  them 
without  inflicting  the  slightest  pain. 

The  other  three  animals  may  be  chloroformed  under 
a  small  bell  glass  on  a  plate. 

Having  killed  either  of  the  first  animals,  make  an  in- 
cision through  the  skin,  from  the  chin  to  the  anus,  and 
reflect  it  on  each  side,  open  the  abdomen  and  remove 
some  of  the  mesentery  and  omen  turn  for  preparation  by 
the  silver  process  ;  open  the  thorax  and  make  an  in- 
cision into  the  heart  and  drain  off  the  blood  before  it 
coagulates.  Before  opening  the  heart  a  small  piece  of 
the  centrum  tendineum  of  the  diaphragm  may  be  care- 
fully cut  out  to  be  prepared  by  the  silver  process.  Then 
open  up  the  thorax  and  abdomen  thoroughly.  Take  out 
the  lungs  with  a  small  portion  of  the  trachea  attached. 
Kemove  the  heart,  open  up  the  ventricles.  Eemove  the 
liver  and  cut  it  into  small  pieces.  Take  out  the  sto- 
mach with  a  piece  of  oesophagus  and  duodenum  at- 
tached, open  it  longitudinally  and  wash  it  in  dilute 


Animal  Tissues  for  Examination.  57 

solution  of  chromic  acid  to  remove  particles  of  food,  &c. ; 
this  must  be  done  gently,  and  it  must  not  be  rubbed. 
Open  the  intestines  with  scissors,  and  wash  in  dilute 
chromic  acid ;  cut  into  short  lengths.  Cut  out  care- 
fully the  ilio-coecal  valve  with  a  portion  of  intestine  on 
either  side.  Eemove  the  kidneys,  and  open  one  longi- 
tudinally, the  other  transversely,  in  two  or  three  places. 
Take  out  the  spleen  and  pancreas  and  cut  them  in 
small  pieces.  Take  out  some  of  the  mesenteric  glands  ; 
remove  carefully  the  uterus  and  ovaries  if  a  female. 
Open  the  scrotum  and  remove  the  testes,  in  many  cases 
these  can  be  got  out  more  easily  by  pushing  them  up 
into  the  abdomen.  Dissect  out  the  penis  and  remove 
it.  Then  cut  round  the  skin  of  the  neck  and  disarticu- 
late the  skull,  taking  care  to  leave  the  remaining  por- 
tions of  the  trachea  and  oesophagus.  Disarticulate  the 
lower  jaw  and  remove  the  tongue  with  the  trachea  and 
oesophagus  ;  cut  off  these  at  the  base  of  the  tongue, 
then  divide  that  organ  longitudinally,  leaving  one  side 
whole,  make  transverse  cuts  in  the  other.  If  large  it 
will  be  necessary  to  make  some  longitudinal  incisions  in 
the  first  for  hardening  with  chromic  acid ;  but  this 
should  be  done  so  as  not  to  interfere  with  making  longi- 
tudinal sections  of  the  whole  of  one  side.  Take  out  the 
submaxillary  glands.  The  eyes  and  brain  will  then 
only  remain,  and  great  care  must  be  exercised  in  taking 
them  out. 

The  best  plan  is  to  remove  the  brain  first ;  for  this 
purpose  take  off  the  covering  of  the  nasal  organ  with  a 
pair  of  bone  forceps,  and  snip  off  pieces  of  bone  from 
the  upper  part  of  the  skull,  taking  care  not  to  touch  the 


58  Dissection  of  Frog. 

brain  underneath,  at  the  same  time  care  must  be  used 
not  to  squeeze  the  eyes  in  holding  the  skull.  When  all 
the  upper  part  of  the  brain  is  laid  bare,  the  bone  should 
be  nipped  through  in  front  of  it  with  the  forceps,  and 
the  base  of  the  skull  divided  ;  by  gently  cutting  away 
the  bone  by  degrees  and  dividing  the  nerves  proceeding 
from  the  brain,  it  can  be  completely  freed  with  the 
cerebellum,  pons,  and  medulla  intact. 

•  It  requires  some  practice  and  care  to  do  this  well, 
but  even  if  the  brain  is  a  little  torn  in  removing  it, 
there  will  still  be  plenty  to  harden  and  make  sections 
of.  The  bone  can  now  be  cut  through,  round,  and  in 
the  orbit  the  optic  nerve  and  recti  muscles  divided,  and 
the  eye  removed  whole. 

Portions  of  nerve  can  be  taken  from  different  parts, 
the  optic,  sciatic,  &c.  Portions  of  muscle  may  also  be 
cut  out.  The  aorta  and  some  of  the  larger  arteries 
should  be  removed  and  cleaned  from  the  surrounding 
tissues. 

DISSECTION  OF  FROG. 

The  whole  eye  may  be  removed  from  a  frog  and 
placed  in  Muller's  fluid,  but  the  cornea  is  required  to 
make  a  gold  preparation. 

Having  killed  a  frog,  wrap  it  in  a  cloth  and  render 
the  eye  tense  with  the  thumb  of  the  left  hand.  Insert 
one  of  the  points  of  a  fine  pair  of  curved  scissors  at  the 
edge  of  the  cornea  and  cut  it  round  carefully,  separate 
it  from  the  rest  of  the  eye,  and  place  it  in  gold  chloride. 

The  mesentery  and  meso-gastrium  may  be  prepared 
by  the  silver  process. 


Dissection  of  Newt  and  Salamander.  59 

DISSECTION  OF  NEWT  AND  SALAMANDEK. 

Kill  a  newt  and  cut  off  the  head,  fix  it  to  the  table 
on  its  back,  by  a  needle  passed  through  the  neck  and 
another  through  the  tail.  Make  a  longitudinal  incision 
down  to  the  anus,  take  up  the  upper  end  of  the 
stomach  with  a  pair  of  forceps,  and  by  carefully  cutting 
away  the  connective  tissue  attaching  it  to  the  cavity, 
the  whole  of  the  contents  of  the  body  can  be  removed 
en  masse. 

Cut  away  the  lungs  and  liver,  which  are  not  wanted. 
Make  an  incision  through  one  side  of  the  stomach  in  its 
whole  length,  and  then  put  the  whole  in  solution  of 
chromate  of  ammonia.  In  Triton  cristatus  there  will 
generally  be  found  in  the  male,  two  testes  on  each  side, 
but  sometimes  three  or  four  ;  some  of  these  may  be  re- 
moved for  the  purpose  of  making  a  fresh  preparation 
of  Spermatozoa. 

Salamander  is  treated  in  the  same  way  for  the  same 
tissues. 


PRACTICAL  HISTOLOGY. 


BLOOD. 

Take  a  small  drop  of  newt's  blood,  cover  and  ex- 
amine :  notice  the  difference  in  shape  and  number 
between  the  coloured  and  white  corpuscles.  Prick 
the  finger  and  examine  human  blood  in  the  same  way. 

AMOEBOID  MOVEMENT. 

A  warm  stage  is  required  to  show  the  amaeboid 
movement  of  the  white  blood  corpuscles. 

With  a  camel's  hair  pencil  apply  a  little  oil  round 
the  edge  of  a  cover  glass,  place  a  small  drop  of  per- 
fectly fresh  newt's  blood  in  the  centre  of  the  glass, 
and  cover  with  another ;  lay  the  preparation  on  the 
warm  stage  over  the  central  hole,  and  apply  the  spirit 
lamp  to  the  wire.  The  thermometer  should  rise  to 
30°  C. 

Select  one  of  the  large  colourless  corpuscles,  and 
sketch  the  different  movements  shown  by  it  at  distinct 
intervals.  Make  a  similar  preparation  of  human  blood, 
and  examine  in  the  same  manner.  The  cover  glass 
must  be  warmed  for  human  blood,  and  the  top  cover 
glass  should  be  touched  on  the  spot  of  blood  coming 
from  the  pricked  finger.  The  coloured  blood  corpuscles 
will  form  rouleaux  if  properly  prepared. 

A  preparation  of  fresh  newt's  blood  should  be  ex- 
amined with  a  high  power  on  the  warm  stage  to  see  the 


Feeding  Blood  Corpuscles.  61 

beautiful  intra-nuclear   and  in tra- cellular  network  of 
fine  fibres  in  the  red  corpuscles. 

FEEDING  BLOOD  COKPUSCLES. 

Eub  up  vermilion  cake  in  f  per  cent,  salt  solution, 
add  a  very  small  drop  of  this  to  the  blood  on  a  slide, 
cover  and  examine  on  the  warm  stage,  paint  a  little 
oil  round  the  edges  to  prevent  evaporation. 

In  the  case  of  newt's  blood  the  thermometer  should 
not  rise  above  30°  C.,  and  in  that  of  human,  not  above 
40°  C.  After  a  time  the  white  blood  corpuscles  will  be 
found  to  have  enclosed  some  of  the  vermilion  granules. 
Newt's  blood  from  the  larger  size  of  the  corpuscles  will 
show  this  best. 

IRRIGATING  BLOOD  CORPUSCLES. 

Make  a  f  per  cent,  saline  solution  of  newt's  blood  on 
a  slide,  cover  and  examine  :  notice  the  red  corpuscles 
gradually  becoming  crenated.  Try  the  effect  of  irriga- 
tion on  this  preparation,  place  a  small  piece  of  filter 
paper  in  contact  with  the  edge  of  the  cover  glass,  and 
with  a  capillary  tube  place  a  small  quantity  of  the  fluid 
to  be  introduced  on  the  opposite  side  ;  as  the  filter 
paper  withdraws  fluid  from  one  side,  the  new  fluid  will 
flow  in  from  the  other.  When  thicker  fluids,  such  as 
glycerine  are  to  be  removed,  a  capillary  tube  is  neces- 
sary on  one  side  to  remove  it  by  suction  with  the  mouth. 

The  fluids  to  be  tried  by  irrigation  are :— Dilute 
acetic  acid ;  distilled  water ;  2  per  cent,  solution  Tannic 
acid;  2  per  cent,  solution  Boracic  acid.  For  their 
effect  the  student  is  referred  to  the  Atlas  of  Histology 
by  Dr.  Klein. 


62  Squamous  Epithelium. 

HJEMIN  CRYSTALS. 

These  are  interesting  as  demonstrating  the  presence 
of  blood,  and  they  are  very  easily  prepared,  and  can 
be  sealed  up  and  kept  as  a  permanent  preparation. 
Glacial  acetic  acid  decomposes  the  blood  pigment,  and 
forms  a  hydrochlorate  of  haematin  in  the  presence  of 
sodium  chloride. 

Place  some  ordinary  table  salt  in  a  watch  glass,  and 
hold  it  over  a  spirit  lamp  until  it  is  thoroughly  dried. 
Take  equal  parts  of  any  dried  blood  and  this  dried 
salt  on  a  slide,  put  on  a  cover  glass,  and  with  a  capil- 
lary tube  run  in  glacial  acetic  acid,  warm  it  gently  over 
a  spirit-lamp  until  a  good  deal  of  the  acid  has  evapo- 
rated, and  then  examine  under  the  microscope.  To 
mount  this  as  a  permanent  preparation,  wash  out  with 
distilled  water  all  the  glacial  acetic  acid,  and  seal  with 
Hollis'  glue.  It  is  better  to  use  some  rather  largish 
masses  of  blood,  or  when  the  acid  is  being  washed  out 
all  the  crystals  will  go  as  well,  the  larger  masses  will 
be  held  by  the  weight  of  the  cover  glass,  and  their 
margins  wi]l  be  found  covered  with  Hsemin  crystals. 

EPITHELIUM. 

SQUAMOUS  EPITHELIUM. 

With  a  blunt  knife  scrape  a  little  saliva  from  the 
back  of  the  tongue  or  inside  of  the  cheek,  cover  and 
examine.  Look  for  squamous  epithelium,  the  so-called 
salivary  corpuscles  showing  Brownian  movements  of 
the  granules  they  contain,  micrococci  and  in  many 
cases  bacteria.  « 


Columnar  Epithelium.  63 

Shed  skin  of  newt.  This  makes  the  best  preparation 
of  surface  epithelium.  Place  a  newt  in  a  glass  jar  of 
water,  and  in  three  or  four  days  it  will  he  found  that 
it  has  shed  the  entire  outer  layer  of  epithelium  as  a  con- 
tinuous skin ;  unroll  it  carefully,  and  cut  into  small 
pieces,  stain  with  logwood  and  mount  in  glycerine. 
The  staining  will  be  facilitated  by  first  placing  it  for 
a  short  time  in  slightly  acidulated  water.  Seal  with 
Hollis'  glue,  and  examine  with  a  low  power.  The 
outlines  of  the  cells  and  their  nuclei  will  be  very  well 
shown. 

Make  a  vertical  section  of  skin  prepared  in  chromic 
acid  mixture  (wherever  this  mixture  is  mentioned,  solu- 
tion No.  1,  p.  11  is  meant).  Stain  in  logwood  and 
mount  in  Canada  balsam. 

Observe  the  different  layers  of  cells,  and  with  a  high 
power  seek  for  the  prickle  cells  of  the  Eete  Malpighi. 
These  are,  however,  better  seen  in  some  epithelial 
Cancers. 

COLUMNAE  EPITHELIUM. 

Take  the  stomach  of  a  newt  or  salamander  prepared 
in  solution  of  chromate  of  ammonia  (page  13),  and  wash 
well  until  no  colour  comes  away,  place  the  whole  in  a 
dilute  solution  of  picro- carmine,  about  15  drops  to  a 
watch  glass  of  distilled  water,  and  let  it  remain  until  it 
has  taken  on  a  deep  red  colour,  then  remove  and  wash 
off  the  excess  of  colouring  matter  ;  scrape  off  a  little  of 
the  surface  material  from  the  inside  of  the  stomach, 
and  tease  out  gently  on  a  slide  in  a  drop  of  glycerin, 
cover  and  examine. 


64  Ciliated  Columnar  Epithelium. 

With  a  high  power  the  network  in  the  cells  and  their 
nuclei  will  be  seen  as  depicted  in  the  Atlas. 

Make  a  longitudinal  section  of  the  large  intestine  of  cat, 
dog  or  rabbit,  prepared  in  chromic  acid  mixture,  stain  with 
logwood,  mount  in  Canada  balsam,  and  examine.  The 
columnar  cells  will  be  seen  in  rows,  some  of  them  having 
become  goblet  shaped,  that  is  distended  by  their  secre- 
tion, and  if  the  animal  has  been  killed  some  time  after 
feeding,  when  digestion  is  going  on,  these  goblet  cells 
will  stain  deeply  with  logwood,  and  the  mucin  will  be 
seen  poured  out  from  the  cell  in  deeply-stained  masses. 
If,  however,  the  animal  has  been  killed  within  a  short 
time  of  feeding,  that  is  before  the  mucigen  has  been 
changed  into  mucin ;  these  goblet  cells  will  not  stain 
with  logwood  but  will  stain  with  anilin  colours. 

CILIATED  COLUMNAR  EPITHELIUM. 

Take  a  portion  of  the  trachea  of  cat,  prepared  in 
2  per  cent,  solution  of  bichromate  of  potash,  wash  well 
in  water,  and  stain  the  mass  in  logwood ;  it  must  be 
put  in  a  strong  solution  and  left  in  for  several  hours ; 
when  deeply  stained  remove  it,  wash  well  until  no  more 
colouring  matter  comes  away.  Then  with  a  small 
knife  scrape  away  a  little  of  the  inner  surface,  and 
place  it  in  a  very  small  drop  of  glycerine  on  a  slide, 
pound  it  with  the  rounded  end  of  a  needle  holder  until 
the  whole  drop  is  seen  to  be  permeated  with  fine  par- 
ticles, and  no  large  ones  are  left ;  place  the  cover  glass 
on  gently  and  allow  the  fluid  to  spread  under  it ;  seal 
and  examine. 


Endothelium.  65 


The  ciliated  cells  will  be  found  isolated  and  can  be 
readily  examined.  Two  other  varieties  of  cells  will  be 
found,  which  are  the  cells  of  the  deeper  layers  and  they 
are  not  ciliated ;  try  with  the  J  to  make  out  the  stria- 
tion  of  the  line  running  across  the  cell  at  the  base  of 
the  cilia ;  a  good  glass  will  show  this  ;  the  T^  oil  im- 
mersion will  show  that  this  striation  is  caused  by  the 
cilia,  which  are  continuous  with  the  longitudinal  stria- 
tion in  the  body  of  the  cell. 

Make  a  transverse  section  of  the  epididymis  of  man 
or  dog  prepared  in  chromic  acid  mixture  and  stained 
with  logwood.  Here  the  ciliated  cells  will  be  beauti- 
fully shown,  the  cilia  being  much  longer  than  in  the 
trachea ;  they  can  be  traced  through  the  striated 
border,  with  a  high  power  in  the  same  manner,  as  in 
the  cells  of  the  trachea. 

ENDOTHELIUM. 

Take  a  portion  of  mesentery  of  cat  and  prepare  by 
the  silver  process.  When  it  has  been  left  long  enough 
in  distilled  water,  stain  it  with  logwood  and  mount  in 
glycerine  ;  seal  and  examine. 

The  nuclei  will  be  well  shown  by  the  logwood  stain, 
but  the  outlines  of  the  cells  will  not  probably  be  very 
distinct  at  first,  but  will  become  more  so  by  the  action 
of  light.  It  will  then  be  seen  that  the  silver  has  been 
deposited  in  the  intercellular  substance  between  each 
cell,  giving  it  a  dark  border.  By  focussing  carefully  a 
second  layer  of  cells  will  be  brought  into  view,  whose 
outlines  do  not  correspond  with  the  first ;  these  are  the 
endothelial  cells  on  the  other  surface  of  the  membrane. 


66  Connective  Tissue  Corpuscles. 

In  some  places  masses  of  small  cells  will  be  found 
deeply  stained,  these  are  germinating  cells. 

Other  serous  membranes  should  be  prepared  by  the 
silver  process,  the  centrum  tendineum  of  the  diaphragm 
makes  a  very  good  preparation,  showing  the  groups  of 
germinating  cells,  and  the  difference  in  shape  of  the 
endothelium  on  each  surface. 

The  lymphatic  capillaries  will  also  be  seen  by  the 
different  shape  of  the  endothelium,  giving  them  the  ap- 
pearance of  trabeculse  running  through  the  membrane. 

A  silvered  preparation  of  the  septum  cisternae  lym- 
phaticae  magnae  should  be  made. 

Eemove  the  viscera  from  a  recently  killed  frog,  and  a 
large  lymph  sac  will  be  found  on  each  side  of  the  spinal 
column  behind  the  stomach,  the  septum  separates  each 
sac  from  the  peritoneal  cavity.  It  may  be  stained  in 
situ  by  pouring  a  little  i  per  cent,  solution  of  nitrate 
of  silver  over  it  and  allowing  it  to  remain  a  few  minutes, 
or  the  septum  may  be  carefully  removed  and  placed  in 
silver  solution.  The  septum  will  show  the  germinating 
cells  round  the  opening  of  the  stomata.  In  all  cases 
where  preparations  of  serous  membrane  are  to  be  made, 
the  animal  should  be  bled  first. 

CONNECTIVE  TISSUE  COEPUSCLES. 

Make  a  gold  preparation  of  Tadpole's  tale. 

This  will  show  the  connective  tissue  corpuscles  very 
well  with  their  branched  processes.  Pigment  cells  are 
also  numerous.  Several  of  these  preparations  should 
be  made,  as  they  show  a  great  many  different  struc- 
tures. 


Tendon.  67 


Make  a  preparation  of  Newt's  mesentery  in  5  per 
cent,  chromate  of  ammonia  (page  13),  and  double  stain 
with  picro-carmine  and  logwood  (page  36).  In  this 
preparation  very  large  branched  corpuscles  will  be  seen 
having  the  hyalin  ground  plate  stained  with  logwood ; 
these  make  most  beautiful  objects  for  examination  with 
high  powers. 

The  corneal  corpuscles  will  be  mentioned  in  another 
place. 

TENDON. 

To  show  the  tendon  cells  which  lie  in  the  interfasci- 
cular  lymph  spaces,  take  a  young  mouse  just  killed  and 
remove  the  skin  of  the  tail,  then  with  the  fore- finger 
nail  separate  two  of  the  caudal  vertebrae  and  forcibly 
remove  the  distal  portion.  Several  white  threads  will 
be  left,  these  are  the  tendons.  Take  a  small  bit  of  one 
of  the  finest  and  place  it  in  slightly  acidulated  water 
for  a  short  time,  then  remove  to  half  per  cent,  gold 
solution,  let  it  remain  about  twenty  minutes.  Then 
place  it  in  distilled  water,  which  must  be  changed  once 
or  twice,  until  it  becomes  a  bronze  colour.  Take  a 
small  bit  and  place  it  in  a  drop  of  glycerine  on  a  slide, 
and  separate  it  into  as  many  fibrils  as  possible.  Cover 
and  examine. 

Take  the  tail  of  a  young  rat  and  prepare  it  in  gold 
chloride  (page  14).  Make  .transverse  sections  and 
double  or  treble  stain  them.  The  tendon  cells  will  be 
seen  darkly  stained,  with  the  gold  lying  between  the 
bundles  of  fibrous  tissue  forming  the  tendons. 

Tendon  should  also  be  examined  in  the  fresh  state, 

F2 


68  White  Fibrous  Tissue. 

by  taking  a  small  portion  from  the  mouse's  tail  and 
mounting  in  salt  solution,  then  irrigating  it  with  very 
dilute  acetic  acid,  and  watching  the  change  that  takes 
place,  as  the  fibrous  tissue  swells  up  and  becomes  in- 
distinct, the  cells  becoming  granular. 

The  acetic  acid  should  be  only  just  sour  to  the  taste. 

After  a  time  the  whole  of  the  fibrous  tissue  will  have 
disappeared,  leaving  a  very  few  elastic  fibres  which  are 
untouched  by  the  acid. 

Take  also  some  of  the  fresh  tendon  and  place  it  in 
logwood  stain,  to  which  a  few  drops  of  glycerine  have 
been  added  ;  let  it  remain  until  deeply  stained.  Tease 
out  small  portions  in  glycerine  on  a  slide ;  the  tendon 
cells  will  be  well  shown  by  this  process. 

ELASTIC  TISSUE. 

Make  a  preparation  of  mesentery  of  frog  and  mount 
in  glycerine  ;  a  very  fine  network  of  elastic  fibre  will  be 
found  throughout  the  whole  structure. 

Take  a  small  slice  of  the  ligamentum  nuchse  of  the 
ox,  which  can  be  readily  procured  from  the  butcher's. 
Place  it  in  dilute  acetic  acid  for  some  little  time,  until 
it  swells  up,  then  tease  a  small  portion  in  a  drop  of 
glycerine  on  a  slide.  Cover  and  examine. 

WHITE  FIBKOUS  TISSUE 

Is  well  shown  in  many  of  the  preparations  of  serous 
membranes.  A  special  preparation  should,  however, 
be  made,  by  hardening  omen  turn  in  1  to  2  per  cent, 
bichromate  of  potash  and  staining  with  logwood;  it 
will  show  the  large  amount  of  fibrous  tissue  present  in 


Cartilage.  69 


a  serous  membrane,  forming  the  greater  part  of  the 
framework. 

White  fibrous  tissue  is  also  well  seen  in  sections  of 
skin ;  also  in  submucous  tissues. 

ADIPOSE  TISSUE. 

Well  seen  in  some  of  the  preparations  of  serous 
membrane,  when  the  fat  cells  lie  thickly  along  the  sides 
of  the  blood  vessels.  Also  seen  in  cutis  vera,  and  in 
many  other  parts.  It  is  not  necessary  to  make  a  spe- 
cial preparation  of  it. 

A  serous  membrane  placed  for  a  short  time  in  dilute 
osmic  acid,  and  mounted  in  glycerine,  will  show  the  fat 
cells  differentiated  from  the  surrounding  tissue,  as  they 
have  all  become  blackened  by  the  action  of  the  osmic 
acid. 

CAETILAGE. 
HYALINE  CARTILAGE. 

The  thin  cartilaginous  expansions  from  the  sternum 
of  the  newt,  prepared  by  the  gold  process,  make  very 
good  specimens  of  hyaline  cartilage.  Thin  sections 
may  be  cut  by  the  microtome,  or  by  hand.  In  this 
preparation  the  lymph  canals  will  be  seen  looking  like 
dark  processes  proceeding  from  the  lacuna,  in  which 
the  cell  lies,  into  the  hyaline  matrix. 

Sections  of  the  nasal  cartilages  of  small  animals, 
growing  bone,  &c.,  will  all  give  good  examples  of  hya- 
line cartilage. 

In  the  fresh  state  the  cells  will  be  seen  to  fill  the 


10  Cartilage. 

lacunae ;     but  in  hardened   specimens   they  have    all 
shrunk,  more  or  less,  leaving  a  space. 

Specimens  of  cartilage  should  also  be  hardened  in 
chromic  acid  mixture,  and  thin  sections  stained  with 
logwood. 

FlBKO-  C  AKTILAGE . 

Make  a  longitudinal  section  of  mouse's  tail,  and 
notice  the  intervertebral  cartilage  and  its  gradual  tran- 
sition into  hyaline  cartilage  on  the  bone. 

The  fibres  will  be  seen  in  various  aspects  as  they 
cross  one  another,  and  several  sections  should  be  ex- 
amined; the  ceUs  will  be  seen  lying  between  the  fibres. 
Make  a  section  of  the  intervertebral  disc  of  sheep  or  ox, 
hardened  in  chromic  acid  mixture,  and  stain  with  log- 
wood. Mount  some  sections  whole  and  tease  out  others 
on  the  slide.  Cover  and  examine.  It  will  be  difficult 
to  make  out  the  fibres  in  some  sections. 

ELASTIC  CARTILAGE. 

This  can  be  well  shown  in  the  lobe  of  the  ear  or  in 
the  epiglottis.  Procure  the  epiglottis  of  a  sheep  and 
harden  it  in  the  chromic  acid  mixture,  cut  sections  and 
stain  them  with  logwood. 

The  ear  of  a  child  prepared  in  the  same  manner,  sec- 
tions cut  and  stained  with  logwood. 

The  ear  lobe  of  a  pig  also  prepared  in  chromic  acid 
mixture  and  stained  with  logwood.  Sections  of  these 
must  be  thin  and  they  must  not  be  hardened  too  much 
or  it  will  be  difficult  to  cut  them. 

The  pig's  ear  when  well  prepared  makes  a  very  use- 
ful specimen,  as  it  shows  a  great  many  tissues. 


Bone.  71 

BONE. 

PREPARING  HARD  BONE. 

Bone  must  be  examined  in  two  forms,  first  in  its  dry 
state,  and  secondly  when  it  has  been  decalcified  or  had 
its  earthy  salts  removed.  In  the  case  of  dry  bone  a  very 
few  sections  will  suffice  as  it  is  a  difficult  and  laborious 
task  to  get  them  well  made. 

The  bone  is  fixed  in  a  vice  and  sections  as  thin  as 
possible  are  cut  with  a  fine  saw,  these  are  rubbed  down 
with  fine  emery  on  a  stone,  and  finally  polished  on  a 
hone ;  they  must  be  well  washed  to  remove  all  debris 
and  are  better  mounted  dry,  as  they  are  apt  to  become 
too  transparent  when  mounted  in  glycerine  or  other 
media. 

DECALCIFYING  BONE. 

It  is  a  very  different  matter  to  make  sections  of  a 
bone  after  the  earthy  salts  have  been  removed,  as  it 
can  be  cut,  as  easily  as  any  other  tissue,  with  the  freez- 
ing microtome  or  razor. 

To  macerate  small  bones,  such  as  mouse  tail,  half  per 
cent,  chromic  acid  will  be  sufficient ;  they  should  not  be 
left  in  too  long.  Larger  bones  must  be  cut  into  small 
pieces  and  placed  in  half  per  cent,  solution  of  chromic 
acid  for  a  week  or  ten  days,  and  then  one-twentieth  of 
the  volume  of  hydrochloric  acid  added  to  the  original 
fluid.  First  harden  then  soften  them,  five  or  six  days 
after  the  addition  of  the  acid  will  be  enough  according 
to  the  size  of  the  bones.  They  should  then  be  tho- 
roughly washed  in  water  for  several  days  or  a  week, 


72  Muscular  Tissue. 


according  to  size,  to  get  rid  of  the  lime  salts,  and  then 
preserved  in  spirit. 

Bone  may  be  macerated  in  a  saturated  solution  of 
picric  acid,  but  it  does  not  act  so  well  as  the  above. 
It  must  be  kept  saturated  by  the  addition  of  fresh  crys- 
tals. 

Make  longitudinal  sections  through  the  head  of  a 
long  bone  such  as  the  femur  of  a  small  kitten,  double 
stain  with  picro- carmine  and  logwood  and  mount  in 
Canada  balsam.  Take  a  portion  of  the  lower  jaw  of  a 
very  young  kitten  near  the  condyle  and  decalcify  it. 
When  prepared  cut  transverse  sections  and  double 
stain  them  with  picro- carmine  and  logwood  and  mount 
in  Canada  balsam.  Sections  also  through  the  carpus 
and  tarsus  of  a  foetal  child  or  kitten  may  be  made  and 
stained  in  the  same  manner. 

The  easiest  specimens  of  growing  bone  to  be  got  are 
from  kittens  just  born,  they  should  be  decalcified  in 
chromic  acid  as  mentioned  before. 

MUSCULAE  TISSUE. 
NON-STKIPED,  STRIPED,  HEART  MUSCLE. 

1.  Non-striped  Muscle.  Make  a  preparation  of  the 
mesentery  of  newt  or  salamander  in  chromate  of 
ammonia,  page  13,  and  mount  in  glycerine.  This 
specimen  shows  better  when  double  stained  with  picro- 
carmine  and  logwood,  page  36. 

Examine  first  with  a  low  power  and  see  the  distri- 
bution of  the  muscle  fibres  through  the  mesentery,  and 
then  with  a  high  power  to  see  the  structure  of  the  in- 


Muscular  Tissue.  73 


dividual  fibres  ;  with  the  one- sixth  the  network  in  the 
nucleus  can  be  seen  but  it  will  require  a  one- eighth 
or  Zeiss'  E  to  make  out  the  fibrils  passing  out  of  the 
ends  of  the  nucleus  into  the  body  of  the  cell,  look  care- 
fully also  for  the  transverse  markings  on  the  cell  and 
the  difference  of  its  diameter  in  some  places,  compare 
their  appearance  with  the  plate  in  the  Atlas  of  histo- 
logy. 

These  fibres  are  very  large  and  give  a  good  idea  of 
the  structure  of  a  non- striped  muscle  fibre. 

In  the  large  area  in  which  this  tissue  is  distributed 
throughout  the  body  of  a  mammalian  animal,  the  in- 
dividual cells  are  very  much  smaller  and  their  struc- 
ture cannot  be  made  out  without  a  high  power, 
especially  as  they  he  very  thickly  together.  In  the 
intestine,  however,  a  thin  section  will  show  a  few 
fibres  running  up  from  the  muscularis  mucosae  to  the 
basement  membrane,  and  in  these  when  well  prepared 
and  stained  the  same  structures  can  be  seen  as  in  the 
much  larger  ones,  in  the  mesentery  of  newt. 

To  see  the  intercellular  substance  by  which  the  mus- 
cle fibres  are  held  together,  take  a  small  portion  cut 
longitudinally  from  the  intestine  and  prepare  it  in 
chromate  of  ammonia,  page  13,  and  stain  in  logwood. 
Cut  longitudinal  sections  so  that  the  circular  muscle 
coat  of  the  intestine  is  cut  transversely,  and  it  will  be 
seen  that  each  fibre  is  separated  from  the  others  by 
a  homogeneous  substance — the  intercellular  cement. 
There  will  be  only  a  few  nuclei  cut  through  and  these 
will  be  seen  deeply  stained. 

2.  Striped  Muscle.      Striped  muscle   is   best   shown 


74  Muscular  Tissue. 

in  one  of  the  large  water  beetles,  Hydrophylus 
piceus.  There  is  another  water  beetle  often  sold 
for  this  purpose  which  does  not  answer  so  well,  the 
Dytiscus  marginalis,  it  may  be  distinguished  by  being 
smaller  and  having  a  yellow  line  round  the  margin  of 
the  upper  surface. 

Fresh  Preparation  of  Muscle  to  show  the  Sarcolemm,a. 
Kill  the  beetle  and  remove  one  of  the  legs,  open  the 
chitinous  covering  and  snip  off  a  bit  of  the  muscle, 
place  it  on  a  slide  in  a  drop  of  distilled  water,  tease  it 
out,  cover  and  examine.  The  sarcolemma  will  be  seen 
in  places  raised  from  the  muscle  substance. 

Irrigate  the  same  preparation  with  dilute  acetic  acid 
and  the  nuclei  of  the  muscle  corpuscles  will  soon  come 
into  view. 

To  make  a  permanent  preparation  stain  a  small  por- 
tion of  the  leg  muscle  with  logwood,  tease  it  out  care- 
fully into  as  fine  fibrils  as  possible  in  a  drop  of  glycerine 
on  a  slide,  cover  and  seal  up  with  Hollis'  glue.  Dissect 
out  some  of  the  muscles  of  the  thorax  and  mount  in  the 
same  way.  Examine  the  muscles  in  a  transverse  sec- 
tion of  the  tongue  of  any  animal  prepared  in  chromic 
acid  mixture  and  double  stained  with  picro- carmine 
and  logwood. 

3.  Heart  Muscle.  Sections  made  from  the  heart  of 
any  small  mammal  will  show  the  peculiarities  of  this 
variety  of  muscle  fibre.  Make  transverse  and  longi- 
tudinal sections,  and  stain  them  with  logwood.  Note 
the  position  of  the  muscle  corpuscles  in  the  transverse 
sections,  and  the  anastomosing  of  the  fibres  in  those 
cut  longitudinally. 


Nervous  Structures.  75 

NEKVOUS   STEUCTUEES. 

MEDULLATED  NERVE  FIBRES. 

Dissect  out  the  sciatic  nerve  of  a  frog  and  stain  for  a 
few  minutes  in  half  per  cent,  solution  of  nitrate  of 
silver,  wash  well  and  expose  to  the  light  in  distilled 
water  until  it  has  become  of  a  brown  colour  ;  cut  small 
portions  and  gently  tease  out  so  as  to  separate  the 
fibrils,  this  must  be  done  carefully  in  a  drop  of  gly- 
cerine on  a  slide  ;  cover  and  examine. 

NODES  OF  EANVIER. 

These  will  be  found  marked  by  black  crosses  in  the 
course  of  the  nerve  fibre,  the  silver  being  heavily  de- 
posited at  the  node  and  penetrating  a  short  distance 
either  way  giving  the  appearance  of  a  cross.  These 
nodes  can  be  seen  in  the  fresh  nerve  or  in  nerve  hard- 
ened and  stained  in  the  ordinary  manner. 

HARDENING  NERVES. 

Take  any  of  the  larger  nerves  from  the  animals  used 
for  material  and  harden  some  in  2  per  cent,  bichromate 
of  potash,  page  13 ;  others  in  chromic  acid  mixture. 
Cut  longitudinal  and  transverse  sections  of  each  of  these 
and  stain  with  logwood,  mount  in  Canada  balsam. 


76  Nervous  Structures. 

NON-MEDULLATED  NERVE  FIBRE. 

This  is  best  seen  in  a  gold  preparation  of  the  tad- 
pole's tail ;  it  will  require  a  high  power  to  trace  the 
finest  fibres,  Zeiss'  E  or  F,  but  the  one-twelfth  oil  im- 
mersion of  Powell  and  Lealand  with  the  binocular  will 
enable  the  student  to  form  a  more  correct  idea  of  the 
structures  he  is  looking  at  and  their  relations  to  one 
another. 

SPINAL  CORD. 

Procure  a  fresh  spinal  cord  of  calf  from  the  butcher 
and  cut  it  into  pieces  corresponding  to  the  different 
regions.  Prepare  one  half  of  each  of  these  pieces  in  a 
2  per  cent,  solution  of  bichromate  of  potash,  page  13, 
and  the  other  half  in  chromic  acid  mixture  ;  those  pre- 
pared in  bichromate  of  potash  may  be  put  in  as  they 
are,  but  those  in  chromic  acid  mixture  must  be  cut  into 
lengths  of  about  half  an  inch.  The  cord  must  be  care- 
fully handled  care  being  taken  not  to  squeeze  it,  and  it 
should  be  cut  with  a  sharp  razor.  Transverse  sections 
of  these  must  then  be  cut  and  stained  with  logwood, 
some  may  be  stained  with  anilin  blue,  page  29,  others 
double  stained  with  carmine  and  indigo- carmine,  page  37. 

Sections  can  be  cut  by  the  microtome,  but  the  razor 
must  be  very  sharp  and  the  cord  well  hardened.  Very 
thin  sections  can  also  be  cut  by  the  hand  and  each 
section  should  be  washed  off  the  razor  into  spirit  as 
it  is  cut.  This  should  not  be  done  with  a  brush  but 
by  dipping  the  razor  into  spirit.  The  cord  prepared  in 
bichromate  of  potash  will  show  the  nerve  structures, 
that  prepared  in  chromic  acid  mixture  the  neuroglia. 


Nervous  Structures.  77 

To  SHEW  THE  LARGE  MULTIPOLAB  NERVE  CELLS  IN  THE 
SPINAL  CORD. 

Cut  a  piece  of  spinal  cord  through  the  middle  longitu- 
dinally, take  one  side  and  holding  it  in  the  hand  cut 
out  with  a  sharp  razor  as  thin  a  slice  as  possible  of  the 
anterior  column,  stain  this  deeply  in  picro- carmine. 
Take  a  small  portion  and  place  it  in  a  drop  of  gly- 
cerine on  a  slide  and  tease  out  very  carefully  with  two 
needles,  this  must  be  done  gently  and  continued  for 
some  time  until  the  whole  piece  is  reduced  to  very 
minute  portions,  hardly  discernible  except  by  their 
colour ;  cover  and  seal  up. 

If  this  process  has  been  carefully  done,  the  large  cells 
will  be  seen  stained  with  picro-carmine  and  completely 
isolated  with  processes  of  different  lengths,  according  to 
the  care  which  has  been  exercised  in  teasing. 

Several  sections  of  the  anterior  column  should  be 
made  and  after  staining  they  should  be  placed  on  a 
slide  and  examined  with  a  low  power ;  those  which  con- 
tain a  large  number  of  multipolar  cells,  will  be  readily 
seen. 

BRAIN. 

Small  brains,  such  as  rabbit's,  harden  very  well  whole 
by  the  spirit  process,  page  13,  they  must  then  be  placed 
in  absolute  alcohol,  before  doing  this  the  different  parts 
should  be  separated,  viz  :  frontal  lobes,  cerebellum, 
pons,  &c. 

After  remaining  in  the  alcohol  a  few  days,  sections 
may  be  cut  of  any  of  the  parts  to  be  examined  and 


78  Nervous  Structures. 

stained  with  logwood,  which  shows  up  the  structural 
elements  better  than  any  other  stain. 

Human  brain  is  best  hardened  in  2  per  cent,  solution 
of  bichromate  of  potash,  page  13  ;  it  must  be  well 
washed  until  no  more  coltor  comes  away,  before  sections 
are  cut.  This  method  is  equally  applicable  to  all  large 
brains. 

Large  sections  of  brain  may  be  cut  with  the  micro- 
tome, but  for  Histological  work  small  sections  will  be 
found  to  show  all  that  is  required.  It  is  a  compara- 
atively  easy  matter  to  cut  a  large  section,  but  to  pass 
this  section  through  all  the  different  re-agents  and 
finally  to  get  it  laid  out  smoothly  on  the  slide,  without 
tearing,  is  a  very  difficult  matter  indeed. 

Bichromate  of  ammonia  (p.  13),  may  be  used  in  the 
same  manner  as  bichromate  of  potash,  for  all  nervous 
structures. 

To  show  the  neuroglia  of  the  brain,  it  must  be  cut  in 
small  pieces  and  hardened  in  the  ordinary  chromic  acid 
mixture. 

PACINIAN  COKPUSCLES. 

These  are  best  seen  in  the  meso-rectum  of  cat,  where 
they  are  visible  to  the  naked  eye  as  oval  bead-like 
bodies.  Cut  out  a  portion  of  the  meso-rectum  and 
spread  it  on  a  flat  piece  of  cork,  fasten  it  at  the  sides 
by  a  few  pins,  and  invert  the  cork  in  a  vessel  containing 
a  2  per  cent,  solution  of  bichromate  of  potash ;  let  it  re- 
main in  this  for  a  few  days,  then  cut  it  into  small  pieces 
and  wash  well,  place  them  in  a  very  dilute  solution  of  log- 
wood, one  drop  to  a  watch  glass  of  distilled  water  will 


Blood  Vessels.  79 


be  enough.  They  must  be  allowed  to  stain  very  gra- 
dually, as  the  logwood  takes  some  time  to  penetrate  the 
capsules.  The  solution  of  logwood  must  be  changed 
several  times  as  it  is  apt  to  become  granular.  It  will 
take  about  48  hours  or  longer,  to  stain  the  corpuscles 
thoroughly.  When  they  have  taken  in  the  colour  suf- 
ficiently, wash  them  well  in  plain  water  and  mount  in 
glycerine. 

Vertical  sections  should  also  be  made  of  the  pad  of 
a  cat's  foot,  hardened  in  chromic  acid  mixture  and 
stained  in  logwood. 

BLOOD  VESSELS. 
CAPILLAKIES. 

Take  the  tail  of  a  half-grown  Tadpole  or  common 
frog,  and  place  it  in  a  5  per  cent,  solution  of  chromate 
of  ammonium  for  24  hours  to  remove  the  epithelium, 
then  wash  well  until  no  colour  comes  away  in  the 
water,  and  double  stain  with  carmine  and  indigo- car- 
mine (page  37).  Mount  in  Canada  balsam. 

By  this  process  the  capillaries  will  be  deeply  stained 
with  carmine,  and  can  be  seen  in  their  natural  con- 
dition. 

Examine  the  capillaries,  in  a  gold  preparation,  of 
a  Tadpole's  tail  stained  with  logwood. 

Either  of  these  preparations  will  show  them  in  pro- 
cess of  development  from  branched  connective  corpus- 
cles. Examine  them  carefully  for  the  nuclei  of  the 
walls,  and  observe  in  many  the  contained  blood  cor- 
puscles. 


80  Blood  Vessels. 


ARTERIES  AND  VEINS. 

Take  the  aorta  of  the  dog  or  cat  and  prepare  either 
in  chromic  acid  mixture  or  spirit  mixture  (page  11). 
Make  longitudinal  and  transverse  sections,  stain  some 
with  logwood  and  double  stain  others  with  pier o- car- 
mine and  logwood  (page  36).  Mount  in  Canada  bal- 
sam. Also  make  transverse  sections  of  the  whole  aorta 
of  rabbit  and  of  smaller  arteries  and  veins  of  the  same 
and  other  animals,  prepared  as  above.  Stain  with 
logwood  and  mount  in  Canada  balsam.  Examine  these 
carefully  to  see  the  different  amount  of  elastic  tissue 
and  non-striped  muscle  in  the  different  arteries. 

Veins  are  prepared  in  the  same  manner  as  arteries, 
but  as  they  are  so  much  slighter  in  structure  it  is  easier 
to  examine  them  in  situ  in  such  sections  as  tongue, 
kidney,  &c. 

ENDOTHELIUM  OF  BLOOD  VESSELS. 

To  examine  the  endothelium  of  a  blood  vessel  it 
should  be  opened  and  then  placed  for  two  or  three 
minutes  in  half  per  cent,  solution  of  nitrate  of  silver, 
and  exposed  to  the  light  in  distilled  water  until  it  has 
become  a  brown  colour.  Then  pin  it  out  on  a  cork 
and  tear  off  with  the  fine  pointed  forceps  thin  strips 
of  the  intima.  Mount  these  in  glycerine. 

To  examine  the  different  coats  of  a  blood  vessel 
separately,  it  must  be  macerated  for  a  few  days  in  a 
2  per  cent,  solution  of  bichromate  of  potash,  well 
washed,  and  strips  torn  off  from  the  different  coats  ; 
these  can  be  then  stained  and  mounted. 


Salivary  Glands — Pancreas.  81 

LYMPHATIC  GLANDS. 

The  lymphatic  glands  of  the  cat  are  very  good  for 
examination,  and  should  be  perfectly  fresh ;  they  are 
best  hardened  in  chromic  acid  mixture. 

Thin  sections  should  be  made  with  the  microtome 
and  stained  with  logwood.  After  washing,  some  of  the 
thinnest  should  be  placed  with  some  water  in  a  test 
tube  and  shaken  for  half  an  hour  or  more,  to  detach 
the  corpuscles  from  the  adenoid  reticulum.  They  must 
be  shaken  steadily  or  they  will  be  knocked  to  pieces. 
Two  or  three  sections  only  should  be  shaken  at  one 
time.  They  are  afterwards  mounted  in  the  usual  man- 
ner in  Canada  balsam. 

It  is  a  good  plan  to  inject  a  solution  of  Berlin  blue 
into  the  lymph  channels  of  a  lymphatic  gland,  to  de- 
monstrate the  passage  of  lymph  through  it.  It  is  done 
by  inserting  the  point  of  a  hypodermic  syringe,  filled 
with  a  solution  of  Berlin  blue,  through  the  capsule 
of  a  fresh  gland,  and  slowly  injecting  the  colouring 
matter ;  the  gland  is  then  prepared  in  the  usual  man- 
ner, and  sections  cut  and  stained  with  logwood. 

THYROID  GLAND. 

This  may  be  prepared  in  precisely  the  same  manner 
as  a  lymphatic  gland. 

SALIVAKY  GLANDS— PANCEEAS. 
May  be  hardened  in   chromic   acid  mixture  or  in 
spirit  mixture  and  care  must  be  taken  that  they  are 
fresh  and  not  over  hardened.     Sections  may  be  cut  by 
the  microtome  or  by  hand  and  stained  in  logwood. 


82  Alimentary  Canal. 

TEETH. 

Sections  of  hard  teeth  are  prepared  in  the  same  way 
as  bone.  Teeth  may  be  decalcified  by  the  same  pro- 
cess as  that  used  for  bone,  page  71.  Good  sections  of 
teeth  in  situ  may  be  made  by  removing  the  lower  jaw  of 
some  small  animal  as  rat  or  mole  and  decalcifying  it, 
after  the  lime  salts  have  been  thoroughly  washed  out, 
it  should  be  soaked  in  gum  for  24  hours  and  then  cut 
with  the  freezing  microtome. 

Sections  may  either  be  mounted  without  staining  in 
Canada  balsam  or  they  may  be  double  stained  with 
picro-carmine  and  logwood. 

ALIMENTAKY  CANAL. 

STOMACH. 

The  stomach  may  be  prepared  in  several  different 
ways. 

1st  Method.  Eemove  and  open  a  fresh  stomach  of 
dog,  cat  or  rabbit,  and  wash  it  slightly  in  dilute  chromic 
acid,  then  place  it  in  the  ordinary  chromic  acid  mixture 
and  proceed  as  described  at  page  11. 

2nd  Method.  To  show  the  peptic  cells  take  fresh 
stomach  and  wash  quickly,  then  plunge  into  pure  me- 
thylated spirit. 

3rd  Method.  Put  the  stomach  in  Muller's  fluid  un- 
washed, for  48  hours.  Then  cut  narrow  strips  of  the 
mucous  membrane  about  half  an  inch  long  by  one 
eighth  of  an  inch  wide  and  wash  these  in  one-tenth  per 
cent,  osmic  acid,  then  place  them  in  half  per  cent. 


Alimentary  Canal.  83 

osmic  acid  from  one  to  two  hours  to  stain,  then  place 
them  in  one- sixth  per  cent,  chromic  acid  and  complete 
the  hardening  in  the  usual  manner.  The  one- sixth 
per  cent,  chromic  acid  is  here  used  without  the  addi- 
tion of  methylated  spirit. 

Some  sections  of  stomach  should  be  taken  from  the 
different  parts,  pylorus,  cardiac  end,  &c.,  and  these 
should  be  stained  in  logwood ;  those  which  are  required 
to  show  the  peptic  cells  should  be  stained  in  anilin  blue. 

Pyloric  end  of  stomach  with  commencement  of  duo- 
denum should  be  hardened  in  chromic  acid  mixture  or 
if  the  whole  stomach  has  been  hardened  a  portion  show- 
ing the  junction  of  these  two  parts  should  be  cut  with 
the  microtome  and  double- stained,  a  few  sections  also 
should  be  stained  with  logwood.  Examine  these  sec- 
tions for  the  gradual  change  in  the  epithelium  as  the 
one  organ  passes  into  the  other. 

DUODENUM 

May  be  hardened  in  chromic  acid  mixture  and  sec- 
tions stained  with  logwood. 

Notice  Brunner  glands  cut  in  different  sections,  also 
goblet  cells  amongst  the  columnar  epithelium  and  the 
fine  non- striped  muscle  fibres  running  up  from  the 
muscularis  mucosae  to  the  basement  membrane. 

ILEUM. 

The  whole  of  the  intestine  may  be  hardened  in 
chromic  acid  mixture  or  in  spirit  mixture,  page  11,  it 
must  not  be  much  handled  and  should  be  first  slightly 
washed  in  very  weak  solution  of  chromic  acid.  Sec- 

G2 


84  Liver. 

tions  are  best  cut  with  the  freezing  microtome  and  may 
be  stained  in  a  great  many  different  ways. 

In  a  section  containing  a  portion  of  Peyer's  glands, 
the  treble  staining  process,  page  39,  may  be  used,  and 
the  result  will  be  very  good  as  the  Peyer's  glands  take 
on  the  green  alone,  without  combining  another  colour 
with  it,  as  all  the  other  elements  in  this  specimen  do, 
so  that  they  are  brought  out  as  brilliant  light  green 
bodies. 

ILIO-CCECAL  VALVE. 

A  section  should  be  made  through  the  ilio-coecal 
valve  with  a  little  of  the  intestine  on  either  side  of  it, 
and  this  should  be  trebly  stained  by  the  process  men- 
tioned at  page  39.  Some  sections  should,  however, 
always  be  stained  with  logwood  to  compare  with  the 
others,  as  although  double  and  treble  staining  differ- 
entiate the  various  tissues,  logwood  brings  out  the 
structural  element  better  than  any  other  stain. 

SOLITARY  GLANDS. 

Sections  should  be  made  through  a  piece  of  large 
intestine  containing  a  solitary  gland  and  this  will  be 
weU  brought  out  by  the  treble  staining  process. 

LIYEE. 

The  liver  may  be  prepared  for  examination  in  three 
ways  : — 

1.  By  the  ordinary  chromic  acid  mixture,  page  11. 

2.  By  dilute  spirit,  page  13. 

3.  By  Muller's  fluid,  page  12. 


Liver.  85 

1.  The  Chromic  Acid  method.     The  liver  must  be  per- 
fectly fresh  and  cut  into  small  pieces  about  half  an  inch 
square,  these  should  be  placed  at  once  in   the   fluid 
without  washing.     A  large  quantity  of  blood  will  exude 
from  them  after  being  in  the  hardening  fluid  a  short 
time,  and  it  will  be  necessary  to  change  it  in  many  cases 
at  the  end  of  12  hours.     It  is  a  good  plan  to  shake  the 
bottle  containing  the  specimen  in  process  of  hardening 
occasionally,  this  must  be  done  gently,  so  as  to  just  alter 
their  position,  and  when  there  is  a  quantity  of  sediment 
at  the  bottom  of  the  bottle,  and  the  fluid  has  lost  its 
yellow  color  and  begins  to  look  muddy,  it  is  time  to 
change  it.      Portions  of  liver  require  changing  a  little 
oftener  at  first  than  other  normal  structures. 

2.  The  spirit  method  is  used  in  the  ordinary  manner 
for  liver,  but   the  dilute  spirit  will  generally  require 
changing  once  before  using  the  pure  methylated  spirit. 

3.  Midler's  Fluid.      This  may  be  used  when  large 
portions  of  the  organ  are  to  be  hardened  and  when 
time  is  no  object. 

When  the  material  is  well  hardened  by  either  of  these 
processes,  beautiful  sections  may  be  cut  with  the  freez- 
ing microtome,  and  they  show  best  when  stained  with 
logwood. 

The  intra-cellular  and  intra-nuclear  network  is  seen 
very  well  in  the  cells  of  the  liver,  and  makes  an  in- 
teresting object  for  a  moderately  high  power.  The 
specimens  should  be  searched  for  bile  ducts  cut  trans- 
versely, looking  like  minute  triangular  openings  between 
the  cells. 


86  Kidney. 

LUNG 

May  be  hardened  in  either  chromic  acid  and  spirit, 
or  Muller's  fluid,  but  to  harden  it  well  the  fluid  must 
be  injected  into  the  lung  through  the  trachea.  This  is 
very  easily  done :  the  lung  having  been  removed  with 
a  portion  of  the  trachea  attached,  an  ordinary  brass 
syringe  with  ivory  nozzle  is  filled  with  the  hardening 
fluid,  the  nozzle  inserted  in  the  trachea,  and  the  lungs 
gently  distended  with  the  fluid ;  when  sufficiently  full, 
the  trachea  is  tied  and  a  weight  attached.  The  lungs 
are  then  placed  in  a  tall  vessel  containing  the  harden- 
ing fluid,  which  is  changed  as  often  as  necessary. 

To  SHOW  THE  EPITHELIUM  OF  THE  ALVEOLI 

Inject  through  the  trachea  a  f  per  cent,  solution  of 
nitrate  of  silver,  and  then  harden  the  lung  by  the  spirit 
process  (page  13),  and  make  horizontal  sections,  these 
must  be  rather  thick  to  get  a  correct  idea  of  the  epithe- 
lium as  lining  a  cavity.  These  specimens  will  show  the 
stomata  between  the  epithelial  cells. 

Lung  is  best  stained  with  logwood.  Sections  should 
be  made  through  a  bronchus  and  the  small  masses  of 
ganglionic  cells  examined,  these  same  sections  may  also 
be  double  or  treble  stained  to  differentiate  the  glands 
of  the  bronchi. 

KIDNEY. 

This  organ  is  very  well  hardened  in  chromic  acid 
mixture. 

Remove  the  kidneys  from  a  freshly  killed   animal, 


Kidney.  87 

take  one  and  divide  it  transversely  into  several  pieces 
and  place  them  in  the  fluid.  The  other  may  be  divided 
longitudinally  by  one  cut,  and  large  kidneys  may  be 
hardened  in  chromic  acid  in  this  way.  Sections 
may  be  cut  by  the  freezing  microtome,  and  are  best 
stained  in  logwood,  or  double  stained  in  carmine  and 
indigo- carmine  (page  37).  To  show  the  minute  struc- 
ture of  the  cells  in  the  collecting  tubes,  fine  striation  of 
epithelium,  &c.,  Heidenhain's  method  is  the  best. 

Cut  the  kidney  into  small  pieces  longitudinally  in  the 
direction  of  the  pyramids,  and  place  them  in  a  5  per 
cent,  solution  of  chromate  of  ammonia,  from  24 — 48 
hours  in  a  stoppered  bottle,  then  wash  for  several  hours 
until  no  more  color  comes  away,  changing  the  water 
several  times,  and  place  in  dilute  and  then  in  strong 
spirit  in  the  ordinary  way  (page  13). 

Sections  should  be  made  both  vertical  and  transverse 
of  the  cervical  and  medullary  portion  of  the  kidney,  as 
well  as  large  sections  longitudinal  and  transverse  of  the 
whole  organ ;  by  this  mean '  the  general  structure  of 
the  organ  and  the  arrangement  of  the  tubes  can  be 
examined,  while  in  the  smaller  sections,  some  of  which 
should  be  mounted  under  thin  covers,  the  minute  struc- 
ture can  be  studied ;  for  this  no  stain  succeeds  so  well 
as  logwood. 

Examine  sections  of  the  cortical  part  for  the  striation 
of  the  epithelial  cells,  and  notice  the  imbrication  in 

some  parts. 

BLADDER — URE  TER  . 

Cut  into  small  pieces  and  harden  in  chromic  acid 
mixture. 


Genital  Organs — Male. 


To  SHOW  GANGLIA  OF  BLADDER. 

Eemove  the  bladder,  empty  it,  and  place  in  \  per  cent, 
solution  of  gold  chloride  2  hours  in  the  dark.  Then 
place  it  in  water  well  acidulated  with  acetic  acid,  until 
it  has  become  swollen  up  to  a  good  size,  then  preserve 
it  in  glycerine. 

The  gold  will  be  seen  deposited  in  small  patches  here 
and  there  on  the  surface,  one  of  these  is  removed  with 
a  pair  of  curved  scissors  and  mounted  in  glycerine. 

GENITAL   OEGANS— MALE. 
TESTIS. 

The  best  hardening  fluid  for  the  testis  is  undoubtedly 
the  chromic  acid  mixture.  The  organ  is  cut  into  small 
pieces,  or  deep  cuts  made  into  it  with  a  sharp  razor, 
according  to  the  size.  It  must  not  be  washed  and 
should  be  handled  as  little  as  possible. 

Sections  are  made  in  different  directions  through  the 
various  parts,  and  it  is  advisable  to  make  some  large 
sections  through  the  corpus  Highinori  and  globus  major 
to  show  the  structure  and  relation  of  the  different 
parts ;  some  of  these  sections  will  show  the  epididy- 
mis  as  well.  These  sections  if  at  all  large  are  very 
difficult  to  mount,  as  they  break  to  pieces  on  the  lifter, 
and  it  is  often  impossible  to  get  them  on  to  the  slide 
whole  ;  in  this  case  the  best  plan  is  to  use  the  cover 
glass  as  a  lifter,  and  clean  it  afterwards,  when  the 
Canada  balsam  has  set. 

Sections  of  testis  are  best  stained  in  logwood,  or 
double  stained  in  picro-carmine  and  logwood  (page  86). 


Genital  Organs — Male.  89 

Some  of  the  thinnest  sections  should  be  mounted 
under  *003  cover  glasses,  as  the  developing  sperma- 
tozoa form  very  interesting  objects  for  the  highest 
powers. 

EPIDIDYMIS  AND  VAS  DEFEKENS 

Are  hardened  in  the  same  way  as  testis,  and  epididy- 
mis  is  generally  hardened  and  cut  with  the  testis. 

Stain  with  logwood. 

Examine  the  columnar  epithelial  cells  of  the  epididy- 
mis  with  very  long  ciliary  processes.  With  a  good 
object  glass  of  moderate  power  these  processes  can  be 
seen  to  be  continuous  with  the  longitudinal  striation  in 
the  body  of  the  cell,  and  can  be  traced  by  careful 
focussing  through  the  striated  line  at  the  margin  of  the 
cells. 

PKOSTATE,  GLANS,  ETC. 

These  can  all  be  hardened  in  chromic  acid  mixture 
in  the  usual  manner.  The  glans  may  be  placed  in  gold 
chloride  for  two  hours  and  then  hardened  in  spirit, 
after  which  longitudinal  sections  will  show  the  nerve 
structures.  The  glans  of  a  small  animal  hardened  in 
this  way  and  cut  longitudinally,  will  show  the  differ- 
ence between  the  epithelium  of  the  mucous  surface  and 
that  in  the  meatus  and  commencement  of  the  urethra  ; 
it  will  also  show  large  bundles  of  medullated  nerves 
arranged  in  a  peculiar  manner,  and  many  other  things 
worth  studying. 


90  Spermatozoa. 


GENITAL   OKGANS— FEMALE. 
UTERUS,  FALLOPIAN  TUBES,  VAGINA. 

Can  all  be  hardened  in  chromic  acid  mixture,  and 
should  not  be  washed  unless  absolutely  necessary. 

To  examine  the  glands  of  the  uterus  it  is  better  to 
use  an  animal  that  has  borne  young. 

These  organs  may  also  be  hardened  whole  in  Muller's 
fluid  or  bichromate  of  potash.  The  sections  are  best 
stained  in  logwood. 

OVARY 

Is  best  hardened  in  chromic  acid  mixture  and  should 
not  be  handled  more  than  is  absolutely  necessary.  The 
whole  organ  may  also  be  hardened  in  Muller's  fluid. 

MAMMARY  GLAND. 

Cut  small  pieces  and  place  in  chromic  acid  mixture, 
when  hardened  stain  with  logwood. 

PLACENTA. 

The  placenta  of  guinea-pig  is  the  best  for  examina- 
tion, and  should  be  taken  a  little  after  half  the  period 
of  gestation  has  passed.  It  may  be  prepared  in  chro- 
mic acid  mixture  or  in  spirit  mixture.  Vertical  sections 
should  be  cut  as  thin  as  possible,  and  stained  with 
logwood. 

SPEBMATOZOA. 

The  living  spermatozoa  of  Triton  cristatus  make  a 
most  beautiful  preparation  and  are  readily  procured. 


Spermatozoa.  91 


Take  a  large  male  newt  which  may  be  known  by  the 
serrated  crest  or  fin  along  the  back,  and  kill  it,  quickly 
remove  the  viscera,  and  the  testes  will  be  found,  gen- 
erally two  or  three  each  side,  as  small  round  bodies 
which  cannot  easily  be  mistaken.  Take  one  of  these  and 
make  a  small  cut  in  it,  remove  some  of  the  milky  fluid 
which  exudes  to  a  slide,  and  add  a  little  salt  solution  or 
distilled  water.  Cover  and  examine.  A  power  about 
i  will  be  required  to  see  the  spiral  filament  well. 

A  large  number  of  spermatozoa  will  be  seen  in  the 
field  making  slight  lashing  movements  with  the  long 
filiform  body,  and  on  closer  examination  the  filament 
will  be  seen  in  rapid  movement ;  this  movement  com- 
mences at  the  elliptical  body  at  the  base  of  the  head, 
and  gives  the  idea  at  first  sight  that  the  filament  is 
being  poured  out  from  it.  After  watching  for  some 
little  time,  the  movement  will  become  slower,  and  it 
can  then  be  seen  that  the  filament  is  attached  to  the 
body  by  a  membrane,  and  that  it  is  waved  rapidly 
from  side  to  side,  by  carefully  watching  it  as  the 
motion  gets  slower  and  nearly  stops  the  -membrane 
connecting  the  filament  to  the  body,  can  be  clearly 
seen. 

To  MAKE  A  PERMANENT  PREPARATION  OF  NEWT'S 
SPERMATOZOA. 

Place  the  testes  in  5  per  cent,  solution  of  chromate  of 
ammonia  for  24  hours ;  wash  until  no  colour  comes 
away  in  distilled  water,  then  divide  one  of  the  testeg 
in  two,  and  taking  one  half  in  a  pair  of  forceps  press 
the  cut  surface  on  a  glass  slide,  a  small  quantity  of 


92  Spermatozoa. 


fluid  will  adhere  to  the  slide,  to  this  add  a  small  drop 
of  glycerine  and  gently  mix  the  two  fluids.  Cover  and 
examine. 

To  STAIN  NEWT'S  SPEKMATOZOA. 

After  having  washed  away  all  traces  of  the  chromate 
of  ammonia,  make  an  incision  into  the  testis  nearly 
dividing  it,  and  place  it  in  undiluted  logwood  stain  for 
an  hour  or  more,  then  wash  away  all  superfluous  stain 
and  mount  in  glycerine.  Spermatozoa  may  be  double 
stained,  but  it  is  a  very  tedious  process,  as  a  little  too 
long  immersion  in  either  stain  spoils  the  whole  pro- 
cess. The  best  way  is,  after  washing  off  the  super- 
fluous logwood  stain,  to  dip  a  portion  of  the  testis  in 
undiluted  spirituous  solution  of  rosein  ;  a  little  must  be 
mounted  to  see  if  the  stain  is  deep  enough,  if  not  it 
must  be  dipped  again. 

It  is  possible  in  this  way  to  get  spermatozoa  of  newt 
or  salamander  with  the  long  pointed  head  stained  with 
rosein,  while  all  the  other  parts  are  stained  with  log- 
wood. 

MAMMALIAN  SPEKMATOZOA. 

To  make  preparations  of  mammalian  spermatozoa, 
a  little  glycerine  is  placed  in  a  watch-glass,  and  one  or 
two  drops  of  absolute  alcohol  added.  A  cut  is  then 
made  into  the  globus  major  of  a  fresh  testicle,  and  a 
little  of  the  fluid  removed  on  the  point  of  a  knife  and 
placed  on  a  slide  ;  a  small  drop  of  the  glycerine  is  then 
mixed  with  it,  it  is  then  covered  and  sealed  with  HolhV 
glue. 


Special  Senses.  93 


HUMAN  SPERMATOZOA. 

A  small  drop  of  semen  is  mixed  with  glycerine,  to 
which  a  little  absolute  alcohol  has  been  added ;  it  is 
then  covered  and  sealed  up.  It  is  best  in  this  case  to 
use  thin  covers  that  have  been  measured,  and  to  note 
their  thickness  on  the  label,  as  the  human  spermato- 
zoon is  so  very  minute  it  requires  the  highest  powers 
to  make  out  the  filament. 

SPECIAL   SENSES. 
INTERNAL  EAR.     COCHLEA. 

The  guinea-pig  is  the  best  animal,  as  the  large  tym- 
panic bulla  is  easily  exposed.  Eemove  the  periosteum 
from  the  bulla,  and  open  it  carefully  with  the  point  of  a 
pair  of  straight  scissors ;  as  soon  as  a  small  opening 
has  been  made  it  can  be  enlarged,  and  the  cochlea  will 
be  at  once  seen  :  as  much  of  the  surrounding  bone 
must  be  removed  as  can  be  done  without  injury  to  the 
parts  required,  and  it  will  be  ready  for  preparation. 

This  may  be  done  in  several  ways,  but  the  two  fol- 
lowing are  the  best. 

1.  Place  it  at  once  in  absolute  alcohol,  and  let  it 
remain  24-48  hours,  then  place  it  in  ^  per  cent,  solu- 
tion of  osmic  acid  for  24  hours.     After  this  place  it 
in  a  half  per  cent,  solution  of  chromic  acid,  to  which 
1-2  drops  of  hydrochloric  acid  have  been  added.     Let 
it  remain  in  this  until  the  bone  is  softened  throughout. 

2.  Place  the  piece  of  bone  in  the  ordinary  chromic 
acid  mixture  for  a  week,  and  then  remove  it  to  £  per 
cent,  chromic  acid,  to  which  1-2  drops  of  hydrochloric 


94  Special  Senses. 


acid  have  been   added.      Kemove   when   the  bone  is 
softened. 

When  the  bone  is  decalcified  by  either  of  these  pro- 
cesses, it  must  be  washed  for  several  days,  to  remove 
the  lime  salt.  It  is  then  ready  for  cutting  sections. 
In  the  case  of  the  internal  ear,  sections  are  much  better 
cut  by  hand,  as  the  fine  structures  such  as  the  rods  of 
Corti  are  quite  disarranged  by  freezing,  and  cutting 
with  a  microtome. 

The  bone  must  now  be  placed  in  gum  solution,  for 
24  hours,  and  then  removed  to  spirit  slightly  diluted 
with  water.  If  the  spirit  is  too  strong,  the  gum  will 
form  a  substance  like  chalk,  and  quite  as  hard ;  this 
can,  however,  be  softened  by  placing  it  in  water.  When 
the  gum  is  sufficiently  hardened  by  the  spirit,  it  can  be 
embedded  in  wax  mass,  and  sections  cut  by  hand  in  the 
ordinary  manner.  These  sections  must  be  very  gently 
handled,  and  should  not  be  lifted  with  a  needle  but  with 
a  fine  camel  hair  pencil.  They  are  best  stained  in  log- 
wood. It  is  a  good  plan  to  stain  the  whole  bone  in 
logwood  before  placing  it  in  the  gum  solution,  but  care 
must  be  taken  that  it  is  not  too  deeply  stained.  The 
undiluted  logwood  should  be  used,  and  it  will  require 
about  6  hours  or  more  to  stain  it  thoroughly. 

Sections  must  be  cut  through  the  semi-circular  canals 
and  also  through  the  cochlea,  and  the  cochlea  should 
be  so  embedded  that  sections  will  be  cut  through  its 
whole  length. 


Eye.  95 

NASAL   OEGAN. 

The  nasal  organ  is  prepared  in  the  same  manner  as 
the  internal  ear,  and  can  be  conveniently  removed  and 
hardened  with  it.  Transverse  sections  should  be  made 
through  the  anterior  part  to  show  the  membrane  of  the 
respiratory  part  and  transverse  sections  further  back, 
and  show  the  olfactory  membrane  with  its  peculiar 
epithelium. 

The  septum  of  the  nose  should  also  be  carefully  re- 
moved from  a  specimen,  and  longitudinal  sections  made 
of  it :  if  these  sections  are  very  carefully  handled,  they 
will  show  both  the  respiratory  and  olfactory  epithelium 
very  well.  They  are  best  stained  in  logwood  and 
mounted  in  Canada  balsam. 

EYE. 

The  eye  may  be  hardened  in  chromic  acid  mixture 
or  in  Muller's  fluid.  It  must  be  removed  without 
squeezing,  and  a  few  incisions  made,  it  can  then  be 
hardened  whole.  When  sufficiently  hard,  divide  the 
eye  longitudinally  with  a  sharp  razor. 

RETINA. 

The  retina  will  be  found  lying  on  the  inside  of  the 
posterior  part  of  the  eye,  from  which  it  may  be  gently 
detached  by  a  spear-shaped  knife,  it  may  be  then  frozen 
and  sections  cut ;  it  is  better,  however,  to  stain  it  first, 
as  thin  sections  are  so  transparent  it  is  difficult  to  see 
them.  When  prepared  in  Muller's  fluid  the  retina  is 


96  Eye. 

very  brittle,  but  in  chromic  acid  mixture  it  is  much 
tougher  and  can  then  be  cut  in  strips  and  several  frozen 
together  in  the  microtome.  Muller's  fluid  shows  the 
nervous  structure  best,  chromic  acid  the  connective 
tissue.  Good  sections  of  the  retina  may  be  obtained 
by  cutting  the  whole  eye  of  the  frog,  and  it  may  be 
double  stained  first. 

To  do  this,  first  place  the  whole  eye  in  a  strong  solu- 
tion of  rosein  until  it  is  deeply  stained,  then  wash  away 
the  superfluous  stain  in  methylated  spirit,  next  place 
the  eye  for  a  short  time  in  strong  solution  of  iodine 
green  and  wash  it  well,  soak  in  gum  solution  and  freeze. 
By  this  means  very  good  sections  can  be  obtained,  the 
granular  layers  having  stained  with  the  iodine  green, 
the  others  with  rosein. 

They  must  be  mounted  in  Canada  balsam  and  should 
not  be  left  long  in  spirit. 

COKNEA. 

The  cornea  may  be  removed  from  an  eye  hardened 
either  in  chromic  acid  mixture  or  in  Muller's  fluid,  and 
sections  made  with  the  freezing  microtome  ;  they  are 
best  stained  with  logwood. 

To  demonstrate  the  Corneal  Corpuscles  and  Nerves. 

Eemove  the  cornea  from  an  animal  just  killed :  this 
is  done  by  cutting  round  the  margin  with  fine  curved 
scissors.  Place  the  cornea  in  i  per  cent,  solution  of 
gold  chloride  in  the  dark.  Let  it  remain  for  from  one 
hour  to  one  hour  and  a  half  for  a  guinea-pig,  an  hour 


Eye.  97 

and  a  half  to  two  hours  for  a  rabbit.  Then  place  it  in 
distilled  water,  which  must  be  changed  once  or  twice, 
for  24-36  hours  exposed  to  the  light,  it  will  then  have 
become  a  violet  colour. 

It  is  now  placed  in  a  mixture  consisting  of 
Pure  glycerine,  1  part. 
Distilled  water,  2  parts. 

Let  it  remain  in  this  for  two  or  three  days  in  the 
dark.  It  is  then  taken  out,  and  gently  washed  and 
placed  in  a  wide  mouthed  vessel  containing  a  filtered 
nearly  saturated  solution  of  tartaric  acid.  As  it  ab- 
sorbs this  liquid,  the  colour  will  become  darker  and  it 
will  sink  to  the  bottom  of  the  vessel.  The  vessel  is 
now  plunged  into  water  at  a  temperature  of  40°  to  50°  C. 
to  such  a  depth  that  the  two  fluids  will  stand  at  the 
same  height. 

Sections  may  now  be  made  with  a  very  sharp  razor 
by  holding  the  cornea  between  the  finger  and  thumb  of 
the  left  hand.  This  requires  great  care,  and  cannot  be 
done  without  a  good  deal  of  practice.  The  sections 
are  mounted  in  glycerine  and  sealed  with  Hollis'  glue. 

Before  cutting  the  sections,  while  the  cornea  is  still 
in  the  distilled  water,  it  is  well  to  pass  a  camel's  hair 
pencil  gently  over  the  surface,  to  remove  the  gold  de- 
posited there. 

IBIS  AND  SCLEROTIC. 

A  portion  of  the  eye  containing  part  of  the  cornea 
sclerotic  and  iris  may  be  cut  out,  and  frozen.  Sections 
of  this  stained  with  logwood,  show  the  junction  of  the 
cornea  and  sclerotic,  and  the  structure  of  the  iris. 

H 


98  Eye. 

LENS. 

In  cutting  sections  of  the  whole  eye  of  frog,  good 
sections  of  the  lens  can  be  obtained.  In  the  whole  eye 
prepared  by  either  of  the  methods  given,  the  lens  will  be 
found  hardened,  when  the  eye  is  opened  ;  and  sections 
may  be  cut  by  the  freezing  microtome,  but  they  gene- 
rally break  up  if  thin ;  enough,  however,  can  be 
obtained  to  enable  the  student  to  examine  into  the 
structure. 


PRACTICAL  PATHOLOGY. 


ON  PREPARING  AND  MOUNTING  PATHOLOGICAL  SPECIMENS. 

VERY  little  need  be  said  about  preparing  pathological 
specimens,  as  most  of  the  processes  already  mentioned 
will  apply  equally  well  to  morbid  tissues.  It  is  often 
necessary  to  find  out  at  once  what  a  tumour  or  new 
growth  consists  of,  and  for  this  purpose  it  is  necessary 
to  examine  the  fresh  tissue.  In  some  cases  this  is 
sufficiently  hard  to  be  frozen  and  sections  made,  while 
in  others  a  small  portion  can  be  teased  out  on  a  slide 
and  examined. 

This  may  give  a  rough  idea  of  what  the  morbid 
growth  is  composed,  but  for  a  thorough  examination 
and  when  it  is  desired  to  keep  preparations  of  any 
disease,  a  complete  process  of  hardening  must  be 
adopted,  and  sections  cut  and  stained  as  in  normal 
tissues,  and  for  this  purpose  it  is  necessary  to  have  the 
material  as  fresh  as  possible. 

To   MAKE   PERMANENT   PREPARATIONS   OF  A  CANCER  IN  A 
SHORT  TIME. 

This  method  may  be  applied  to  most  of  the  sarco- 
mata and  carcinomata,  and  is  valuable,  as  it  can  be 
used  on  portions  of  morbid  tissue  excised  from  the  liv- 
ing body.  Snip  off  a  small  portion  of  any  morbid 
growth,  such  as  cancer,  with  a  pair  of  curved  scissors. 

H2 


100  Morbid  Growths. 


Place  it  in  a  mixture  of  dilate  spirit  (page  13)  for  12 
hours,  then  remove  it  to  pure  methylated  spirit  for  12 
hours  and  finally  to  absolute  alcohol  for  12  hours.  It 
will  then  in  all  probability  be  fit  to  cut  sections  from. 
Imbed  in  wax  mass  and  cut  some  sections  by  hand, 
stain  with  logwood  and  mount  in  Canada  balsam. 

There  are  some  morbid  growths,  such  as  medullary 
carcinoma,  which  cannot  be  dealt  with  in  this  way,  and 
they  must  be  hardened  in  the  chromic  acid  mixture  in 
the  usual  manner. 

In  all  morbid  growths  where  there  is  a  large  epi- 
thelial element,  they  can  be  best  hardened  in  spirit 
mixture.  When  large  masses  are  to  be  hardened, 
Muller's  fluid  or  2  per  cent,  bichromate  of  potash  are 
necessary,  and  must  be  used  in  the  manner  described 
for  normal  tissue  at  page  12. 

It  is  quite  as  important  that  pathological  specimens 
should  be  properly  hardened  as  normal  tissues,  but 
how  seldom  is  this  done.  In  the  first  place  it  is  diffi- 
cult to  get  the  morbid  tissues  fresh  enough,  and  yet 
they  are  often  put  on  one  side  or  at  most  placed  in  the 
lump  in  a  very  small  quantity  of  methylated  spirit  for 
some  time  before  being  hardened,  and  it  is  expected 
that  good  sections  can  then  be  prepared  from  them. 

Nothing  is  more  erroneous  than  this  idea ;  the  sub- 
ject has  been  probably  dead  24  hours  at  the  least  when 
the  post  mortem  is  made,  often  longer,  and  in  summer 
especially,  this  means  utter  ruin  to  such  organs  as  the 
spleen.  How  important  is  it  therefore  that  such  organs 
as  are  fresh  should  be  put  in  the  hardening  medium  at 
once.  For  this  purpose  a  wide-mouthed  bottle  of  chro- 


Morbid  Growths.  101 


mic  acid  mixture  should  be  taken  to  every  post  mortem 
examination,  and  small  bits  of  any  organ  that  may 
seem  interesting  on  any  account  may  be  put  in.  A 
small  paper  label  may  be  tied  on  and  they  can  be 
separated  afterwards. 

Almost  every  morbid  growth  can  be  hardened  in  the 
chromic  acid  mixture  (page  11),  in  the  same  manner 
as  normal  tissues.  Brain  and  spinal  cord,  however, 
are  better  prepared  in  2  per  cent,  solution  of  bichro- 
mate of  potash  (page  13). 

ON  DOUBLE  AND  TREBLE  STAINING  MOBBID  GEOWTHS. 

Some  very  good  results  will  be  obtained  if  the  differ- 
ent staining  processes  mentioned,  as  well  as  any  others 
that  may  suggest  themselves,  are  tried  on  different 
morbid  growths. 

For  instance,  well  hardened  sections  of  rodent  ulcer 
and  epithelioma  may  be  stained  by  the  indigo- carmine 
process  (page  37)  and  carefully  compared.  Other  sec- 
tions of  the  same  material  should  be  then  stained  with 
rosein  and  iodine  green  (see  treble  staining)  and  again 
compared.  In  this  way,  some  definite  result  may  be 
worked  out,  which  by  using  other  specimens  of  the 
same  disease  may  be  confirmed. 

LAUGHS  SECTIONS  OF  PATHOLOGICAL  SPECIMENS* 

If  a  large  section  is  wanted  of  any  morbid  growth  to 
show  the  distinction  between  the  healthy  and  diseased 
parts,  such  as  a  section  through  a  cancer  and  the  side 
of  the  uterus  from  which  it  is  growing,  it  is  better  to 


102  Hydatids. 


cut  a  moderately  thick  slice,  say  about  a  quarter  of  an 
inch  thick,  and  harden  it  in  chromic  acid  mixture,  than 
to  harden  the  whole  mass  in  spirit,  as  it  will  be  found 
a  very  difficult  matter  to  cut  large  sections  of  spirit 
hardened  material ;  they  become  so  hard  that  they 
make  the  knife  jump,  and  the  section  is  consequently 
uneven. 

AMYLOID  DEGENEKATION 

Is  best  hardened  in  chromic  acid  mixture,  and  in 
kidney  especially  is  very  well  shown  by  this  method. 
To  show  the  amyloid  substance  well  it  must  be  stained 
a  different  colour  to  the  surrounding  tissue,  and  this 
may  be  done  by  several  of  the  double  staining  pro- 
cesses ;  as  the  anilin  dyes  seem  to  have  a  special  af- 
finity for  amyloid  degeneration,  the  following  should 
be  tried : 

Carmine  and  indigo  carmine  (page  87). 
Eosein  and  iodine  green. 
Eosin  and  anilin  blue. 

and  many  other  combinations. 

HYDATIDS. 

To  make  a  preparation  of  hydatid  cysts,  take  a  por- 
tion of  the  wall  of  a  large  cyst  and  scrape  off  some  of 
the  gelatinous  matter  adhering  to  it.  Place  a  little  of 
this  on  a  slide  and  tease  it  gently  in  a  drop  of  glycerine, 
cover  and  examine.  If  there  are  any  small  cysts  show- 
ing booklets,  &c.,  well,  seal  it  up  with  HolhV  glue. 


Morbid  Growth.  103 


SHORT  HISTORY  OF  THE  MANNER  IN  WHICH  A  PORTION  OF 
MORBID  GROWTH  is  PREPARED  BY  THE  CHROMIC  ACID 
METHOD. 

1st  day.     Small  pieces  placed  in  chromic  acid  mix- 
ture (page  11). 

2nd  day.     Fluid  changed. 

5th  day.     Fluid  changed. 

8th  day.     Fluid  changed. 

9th  day.     Spirit  mixture  (page  13). 

10th  day.    Pure  methylated  spirit. 

14th  day.     Plain  water. 

15th  day.     Mucilage. 

16th  day.     Section  cut,  stained,  and  mounted. 


INDEX. 


Absolute  alcohol,  49 

Acetic  acid,  27,  36,  68 

Achromatic  condenser,  8 

Adenoid  reticulum,  81 

tissue,  69 

Air  bubbles,  51 

Alimentary  canal,  82 

Amoeboid  movement,  60 

Amyloid  degeneration  102 

Anilin  blue,  39,  83 

red,  32 

violet,  32 

dyes,  28 

list  of,  22 

soluble  in  spirit,  31 

soluble  in  water,  28 

Anthra-purpurin,  25 

Arteries,  80 

Aurine,  33 


Beetle,  74 
Berlin  blue,  81 
Bicarbonate  of  soda,  24,  37 
Bichromate  of  ammonia,  13 

— potash;  13,  68 

Binocular  microscope,  9 
Bismarck  brown,  31 
Bladder,  87 
Blood,  60 

vessels,  79 

Bone,  71,  72,  94 

decalcifying,  71 

Boracic  acid,  61 
Brain,  56,  77,  101 

human,  73 

Bronchus,  86 
Brunner's  glands,  83 


Cabinet,  52 
Canada  balsam,  48 
Capillaries,  78 
Carmine,  24,  37 
Cartilage,  69 


Cells,  tendon,  67 

Cerebellum,  77 

China  blue,  28 

Chloride  of  gold,  14,  42 

Chloroform,  49 

Chromate  of  ammonia,  13 

Chromic  acid,  11,  24 

Cilia,  65 

Ciliated  epithelium,  64 

Citranine,  33 

Cleaning  cover  glasses,  45 

slides,  44 

Cochlea,  93. 

methods  of  hardening, 

93 

Columnar  epithelium,  63 

Condenser,  8 

stand,  8 

Connective  tissue  corpuscles,  66, 
79 

Cornea,  96 

Corneal  corpuscles,  96 

Corpus  Highmori,  88 

Cover  glasses,  44 

Cutis  vera,  69 

Cutting  sections,  16,  17 

with    micro- 
tome, 19. 


Dahlia,  30 
Dammar  varnish,  48 
Decalcifying  bone,  14,  42 
Dilute  spirit,  13 
Distilled  water,  20,  23 
Double  staining,  34,  36,  37 
Duodenum,  83 
Dytiscus  marginalis,  74 

Ear,  70 

Elastic  tissue,  68 
Endothelium,  65,  80 
Eosin,  26,  39 
Epididymis,  65,  88 


106 


Index. 


Epiglottis,  70 
Epithelioma,  101 
Epithelium,  62 
Epithelial  cells,  striation  of,  87 
Eye,  58,  95 
Eye-piece,  7 

Fallopian  tubes,  90 
Feeding  blood  corpusles,  61 
Fibro-cartilage,  70 
Freezing  microtome,  18 
—  mixture,  19 

Labels,  2 
Lamp,  8 
Large  slides,  54 
Lens,  98 
Lifter,  50 
Ligamentum  nuchse,  68 
List  of  apparatus,  3 

Liver,  84 
Logwood  stain,  23,  37 
Lung,  86 

Lymphatic  gland,  81 

Malachite  green,  30 
Mammary  gland,  90 
Measuring  cover  glasses,  44 
Medullary  carcinoma,  100 
Medullated  nerves,  89 
Mesentery,  27,  37,  58,  68 
Meso-rectum,  78 
Methyl-  a  nilin  violet,  30 
Microscope,  4 
Microtome,  18 
Mounting,  50 
fluids,  46 

Frog,  58 

Ganglia  of  bladder,  87 
Germinating  cells,  66 
Glans,  89 
Glass  capsules,  17 
Globus  major,  88 
Glycerine,  46 
Gold  chloride,  88 
Gauge  for  cover  glasses,  45 
Guinea  pig,  90,  93 
Gum  solution,  94 

Hardening,  11 
Hsemin,  62 
Heart,  56 

f1  yj  5I^°r^gn^' 

large  sections,  53 
Mouse  tail,  14,  43,  68,  71 
Mucilage,  20 
Muller's  fluid,  3,  12,  85 
Multipolar  nerve  cells,  77 
Muscular  tissue,  74 
Muscularis  mucosse,  73 
Muscle  corpuscles,  74,  75 

Nasal  organ,  95 
Nerves,  58,  75 

High  powers,  6 
Hollow  ground  razor,  17 
Hydatids,  ]02 
Hydrochloric  acid,  37,  71 
Hydrophylus  piceus,  74 
Hypodermic  syringe,  81 

Ileum,  83 
Ilio-coecal  valve,  84 
Illumination,  8 
Imbedding,  16 

Indigo-carmine,  25,  37 
Intestine,  56,  73,  83 
Intercellular  substance,  73 
Internal  ear,  93,  94 
Intima,  80 
Intr  a-  cellular  network,  85 
Iodine  green,  30,  39 
Iris,  97 

Kidney,  57,  86 

Neuroglia,  76,  78 
Newt,  91 
Nodes  of  Ranvier,  75 
Non-striped  muscle,  72 

Object  glass,  5 
Oil  immersion  lenses,  6,  10,  76 
Olfactory  organ,  95 
Umentum,  68 
Osmic  Acid,  15,  69,  83,  95 
Ovary,  90 

Killing  animals,  56 

Index. 


107 


Pacinian  corpuscles,  78 
Pad  of  foot,  79 
Pancreas,  81 
Pathology,  99 
Penis,  57 
Peyer's  glands,  39,  84 
Picric  acid,  14,  27,  72 
Picro-  carmine,  27,  36,  39 
Placenta,  90 
Pons,  77 
Preparing  cancer,  99 
morbid  specimens,  103 
Prickle  cells,  63 
Prostate,  89 
Pure  opal  blue,  33 
soluble  blue,  29 
Purpurine,  25 
Pylorus,  83 

Reagents,  3 
Eespiratory  epithelium,  95 
Retina,  21,  95 
Eodent  ulcer,  101 
Rods  of  Corti,  94 
Rosanilin,  30 
Rosein,  32 

Safranine,  29,  39 
Salamander,  59 
Salivary  gland,  81 
Sarcolemma,  74 
Sclerotic,  97 
Sealing  preparations,  48 
Selective  stains,  34 
Semicircular  canals,  94 
Serous  membrane,  69 
Serge  blue,  29 
Silver  process,  65 
Skin,  27 

Slides,  44 
Soluble  anilin  blue,  28 
Solitary  glands,  84 
Spermatozoa,  59,  89,  90 

Spiller's  purple,  31 
Spinal  cord,  28,  76,  101 
Staining,  22 
Stand  condenser,  8 
Stomach,  28,  56,  59,  82 
Striped  muscle,  73 

Tadpole's  tail,  14,  66,  74,  76 
Tannic  acid,  61 
Teeth,  82 
Tendon,  67 
.  ~Dna   an 

Testis,  57,  59,  88 

—              -  nf-no-ctrf     O1 

Thin  slides,  55 
Thyroid  gland,  81 
Treble  staining,  27,  39 
Triton  cristatus,  90 
Tubuli  seminiferi,  37 
Tympanic  bulla,  93 
Tyrian  blue,  29 

Urethra,  89 
Ureter,  87 
Uterus,  90 

Vas  deferens,  89 
Vagina,  90 
Veins,  80 

Warm  stage,  60 
Wax  and  oil  mixture,  16 
White  fibrous  tissue,  68 

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